CN101850108A - Methods and pharmaceutical compositions for healing wounds - Google Patents

Abstract

A pharmaceutical composition for inducing or accelerating a healing process of a damaged skin or skin wound, the pharmaceutical composition comprises, as a n active ingredient, a therapeutically effective amount of insulin and at leas t one additional agent acting in synergy with said insulin, and a pharmaceutically acceptable carrier being designed for topical application o f the pharmaceutical composition. A method of inducing or accelerating a heali ng process of a damaged skin or skin wound, the method comprises administering to the damaged skin or skin wound a therapeutically effective amount of insulin and at least one additional agent acting in synergy with said insulin to induce or accelerate the healing process of the damaged skin or the skin wound.

Description

The method and the pharmaceutical composition that are used for healing wounds

The application is dividing an application of following application: July 15 2004 applying date, application number 200480026529.5 (PCT/IL2004/000640), title " method and the pharmaceutical composition that are used for healing wounds ".

Invention field

Therefore and the method and the pharmaceutical composition of the agglutination of acceleration of wound (wounds) the present invention relates to a kind of be used to induce and/or quicken cell proliferation and/or cell differentiation and.More specifically, the present invention relates to serine/threonine protein kitase through regulating, be also referred to as expression and/or the activation of PKC, for example cause, thereby be used to induce and/or quicken the purposes of the agglutination of cell proliferation and/or cell differentiation and/or cell migration acceleration of wound by film transportation and activation.According to instruction of the present invention, the adjusting of this expression can realize by following effects: (i) transform the wound cell with the PKC expression construct; (ii) transform the wound cell with cis acting element, this element will be inserted into the endogenous PKC upstream region of gene of wound cell close position; (iii) give expression and/or the activation of insulin to induce PKC in the wound cell; (iv) transform the wound cell with the insulin expression construct, the expression and/or activatory the going up that can be used as PKC with excretory insulin of the expression of its generation are adjusted; (v) transform the wound cell with cis acting element, this element will be inserted into the endogenous insulin gene of wound cell upstream close position, be expressed the expression and/or the activatory upward adjustment that can be used as PKC with excretory insulin; (vi) in wound, implant insulin secretory cell; (vii) use trans acting factor, for example PDX1 transforms the wound cell, and to induce endogenous insulin to produce and secretion, described insulin can be used as expression and/or activatory the going up of PKC and adjusts; And (viii) use the PKC regulator to wound.

The present invention as any said method realize, the enforcement of also can exsomatizing is to produce skin graft.

Background technology

The topmost purpose of trauma care is to make wound closure.Open skin trauma has been represented the main wound of a class, comprises burn, neurogenic ulcer, decubital ulcer, venous stasis ulcer, and diabetic ulcer.

Open skin trauma through such process healing, comprises six staples: (i) inflammation usually; (ii) fibroblast proliferation; (iii) vascular proliferation; (iv) connective tissue is synthetic; (v) form epithelium; (vi) wound is shunk.When these key elements, no matter be independent or as a whole, when can not correctly play a role, then destroyed the healing of wound.There is multiple factor can influence wound healing, comprises malnutrition, infect, pharmacological agents (for example D actinomycin D and steroid), advanced age and diabetes [referring to Hunt andGoodson, Current Surgical Diagnosis﹠amp; Treatment (Way; Appleton﹠amp; Lange), pp.86-98 (1988)].

With regard to diabetes, the feature of diabetes just is that insulin signaling conduction (signaling) sustains damage, and plasma glucose raises, and the susceptibility that chronic complication takes place in several particular organizations.In the chronic complicating diseases of all diabetes, the impaired wound healing that causes foot to fester is that research is minimum.Yet the skin ulcer of diabetics has spent a large amount of manpower and financial resources (29,30).And, foot festers and lower limb then to block be the reason (30-33) that modal diabetics is in hospital.In diabetes, wound healing process is damaged, and the wound of healing is characterised in that wound intensity reduces.The defective of tissue repair is relevant with several factors, comprises neuropathy, angiopathy and infection.But, still do not understand other mechanism, by this mechanism, the diabetic disease states relevant with the insulin signaling conduction abnormalities destroyed the healing of wound and changed skin physiology.

But the surgical operation wound healing afterwards that ubiquitous problem is body parts is performed the operation successfully the disunion of wound otch in addition.

Skin is stratified flaky epithelium, is wherein growing and the cell that breaks up is separated by strictness.In physiological status, propagation is only limited to the basal cell that is attached on the basement membrane.Differentiation is a kind of steric course, and wherein basal cell loses and the adhering to of basement membrane, and it is synthetic and experience a series of morphologic and biochemical variations to stop DNA.Final maturing step is to produce horny layer, and it forms the protection barrier (1,2) of skin.The ability relevant (3) that observed variation the earliest and basal cell separate and move from basement membrane when basal cell begins to break up.The agglutination of wound also has similar variation, and wherein cell migration is to wound site, and multiplication capacity also increases.These processes are reconstructions of skin layer and induce the correct differentiation of epidermal area necessary.

The exploitation of mice and people's keratinocyte culture systems greatly facilitates the analysis (2,4) to epidermal growth and differentiation regulatory mechanism.External, keratinocyte can remain the substrate proliferative cell with high speed of growth.And following epidermis cooked mode in vivo can be in external evoked differentiation.Early stage incident comprises forfeiture (3,5) and the selectivity forfeiture of alpha 6 beta 4 integrin and the selectivity that the adheres to forfeiture of cell and stromatin of hemi desmosome component.This shows that the variation of integrin expression is the early stage incident in the keratinocyte differentiation.The forfeiture of early stage hemi desmosome contact (hemidesmosomal contact) causes the basad layer of keratinocyte to go up migration, and with the keratinocyte of cultivating in and the inducing of keratin 1 (K1) relevant (1,3,6) in the skin.To the downward modulation of the expression of the further differentiation of granular layer phenotype and β 1 and beta 4 integrin, relevant with the forfeiture of adhering to potentiality of all stromatins, be exactly the formation and the cell death of hornification coating then.Noble cells finally comes off from culture dish becomes ripe squama (2,7).This vitro differentiation process is that strictness is followed body endepidermis cooked mode and carried out.

The contribution of Protein kinase C (ProteinKinase C) approach has been given prominence in biological research for keratinocyte recently, and it regulates skin proliferation and differentiation.The Protein kinase C of serine-threonine kinase (PKC) family all plays important regulatory role (8,9) in multiple biological phenomenon.PKC family is made of at least 12 independent isotypes (isoform), and it belongs to 3 different classifications: (i) traditional isotype (α, β 1, β 2, γ), the Ca that it is discharged in born of the same parents by phospholipase C ²⁺, Buddhist ripple ester and diacylglycerol activate; (ii) new isotype (δ, ε, η, θ), it also can be activated by Buddhist ripple ester and diacylglycerol, but can not be by Ca ²⁺Activate; (iii) family atypical (ζ, λ, τ) member, it can not be by Ca ²⁺, Buddhist ripple ester or diacylglycerol activate.

When being activated, most of but not all isotype is considered to and can moves on the plasma membrane from Cytoplasm.The type of isotype has nothing in common with each other in different tissues with distribution pattern, also can change with phenotype.Because the importance of PKC in the cell terminal point of multiple hormonal action, its 26S Proteasome Structure and Function has been illustrated in existing multinomial research.Five kinds of PKC isotype-α, δ, ε, η and ζ in the skin have been discerned in vivo with in the culture.The recent PKC signal transduction path that studies show that is the interior regulator (10,11) of main born of the same parents of differentiation reaction.And pharmacology's activator of PKC is in the body and the strong derivant (4,12) of external keratinocyte differentiation, and pkc inhibitor can prevent to break up the expression (10) of mark.

When design was of the present invention, crossing expression and/or activating of conjecture PKC isotype may help the accelerated wound healing process.Owing to be difficult in primary cell, introduce exogenous gene with traditional method effectively, thereby the research of the effect of each PKC isotype in skin cell proliferation and/or differentiation be restricted.Vital stage is short, exists differentiation may and can not be separated to stable transformant and all makes exogenous gene can not effectively transduce into former generation Skin Cell.

Description of the Prior Art the potential application of insulin as the therapeutic agent of wound healing.United States Patent (USP) 5,591,709,5,461,030 and 5,145,679 have described insulin have been locally applied to wound to promote wound healing.But these patents are described all to be that insulin and glucose combination are used, because the function of insulin is the absorption that increases glucose, thereby promotes wound healing.

U.S. Patent application 09/748,466 and International Patent Application PCT/US98/21794 have described and have been locally applied to skin to promote the compositions that contains insulin of the skin injury that skin health or treatment are more shallow.But these patent applications not instruction make insulinize chronic, II degree (Grade II) or darker wound.

International Patent Application PCT/US01/10245 has described cyanoacrylate polymer sealant and insulin or silver is used in combination so that wound healing.But, in this application, both do not had instruction not hint that insulin and another kind of bioactive agents are used in combination expression and/or the activation that can regulate PKC yet.

International Patent Application PCT/US85/00695 has described the local application insulin with the treatment diabetes.But this patent application not instruction makes insulinize and the irrelevant wound of diabetes.

International Patent Application PCT/US92/03086 has described the treatment microemulsion formulation that can contain insulin.But its disclosed content is not instructed and is used insulin preparation to make wound healing.

United States Patent (USP) 4,673,649 and 4,940,660 have described the compositions that is used for people's keratinocyte and the external clone growth of epidermis cell, wherein contain epidermal growth factor and insulin.These patents have been instructed insulin have been used for the cultured skin cells whose development, and described cell can be used for transplanting.But these patents are not instructed insulin are used for wound in vivo.

The above-mentioned prior art list of references of quoting is not instructed or is hinted expression and/or the activation that insulin is used to regulate PKC, thus the agglutination of acceleration of wound.And prior art is not instructed or is hinted and uses nucleic acid construct or gene transformation technology to provide insulin to wound, thus the agglutination of acceleration of wound.

Generally have recognized the need at present, and also need the new method of energy accelerated wound healing correlated process for the purpose of facility.In addition, also generally have recognized the need to, and for the purpose of facility also needs recombination to be inserted into effective ways in the Skin Cell, it will speed up cell proliferation and/or atomization and wound healing.

Summary of the invention

When enforcement was of the present invention, the inventor found that insulin is administered to site of injury separately can produce for example excessive angiogenesis of bad side effect, inflammation, epithelial hyperplasia and cicatrization (embodiment 23 of the embodiment part that sees below).The further discovery of the inventor used the side effect that can effectively avoid insulin to cause with insulin and one or more expression and/or the active agent combination that can regulate PKC in being settled in the cell of wound site (cells colonizing thewound area), and simultaneously remarkable accelerated wound healing process.

The invention provides efficient treatment wound and do not have the new method and the compositions of adverse side effect, comprise to wound site expression that the insulin of effective dose and/or other regulate PKC in can the cell in wound site and/or active and have the process of synergistic reagent with accelerated wound healing with islets of langerhans is provided.

Therefore, according to an aspect of the present invention, the method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided, and described method comprises to the adjusting PKC generation of the skin of damage or skin trauma administering therapeutic effective dose and/or the step of the activatory reagent of PKC.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, at least a the adjustings PKC generation and/or the active reagent of treatment effective dose; And pharmaceutically acceptable carrier.

According to another aspect of the present invention, the method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided, described method comprises the step that have synergistic other reagent to the skin of damage or the insulin of skin trauma administering therapeutic effective dose and at least a and islets of langerhans, with the skin of inducing or quickening to damage or the agglutination of skin trauma.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, the insulin of treatment effective dose, at least a can with synergistic other reagent of insulin, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

The method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided according to another aspect of the present invention, described method comprises the step to the insulin of the skin of damage or the skin trauma treatment effective dose of using single dose, thereby induces or the skin that quickens to damage or the agglutination of skin trauma.

The pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided according to another aspect of the present invention, described pharmaceutical composition comprises, as active component, the insulin of the single dose unit of the agglutination of the selected skin that can induce or quicken to damage or skin trauma, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

A kind of method of inducing or quickening the agglutination of outmoded skin trauma (old skin wound) is provided according to another aspect of the present invention, described method comprises the step of insulin of using the treatment effective dose of single dose to outmoded skin trauma, thereby induces or quicken the agglutination of outmoded skin trauma.

According to another aspect of the present invention, the method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided, described method comprises the step to the skin or the insulin secretory cell of skin trauma implanted treatment effective dose of damage, thereby induces or the skin that quickens to damage or the agglutination of skin trauma.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as the insulin secretory cell of active component, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

According to another aspect of the present invention, the method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided, the cell that described method comprises the skin that transforms damage or skin trauma to be producing and the step of excreting insulin, thereby induces or the skin that quickens to damage or the agglutination of skin trauma.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, be designed for the skin that transforms damage or skin trauma place cell producing and the nucleic acid construct of excreting insulin, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

According to another aspect of the present invention, the method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided, described method comprises the skin that transforms damage or skin trauma place cell producing the step of Protein kinase C, thereby induces or the skin that quickens to damage or the agglutination of skin trauma.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, be designed for the skin that transforms damage or skin trauma place cell producing the nucleic acid construct of Protein kinase C, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

According to another aspect of the present invention, the method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided, described method comprises the step to the skin or the PKC activator of skin trauma administering therapeutic effective dose of damage, thereby induces or the skin that quickens to damage or the agglutination of skin trauma.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, the PKC activator of treatment effective dose, with the skin of inducing or quickening to damage or the agglutination of skin trauma, and pharmaceutically acceptable carrier.

According to another aspect of the present invention, provide the method for the agglutination of a kind of skin of inducing or quickening to damage or skin trauma, described method comprises the step to the copolymer-1 of the skin of damage or skin trauma administering therapeutic effective dose.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, the copolymer-1 of treatment effective dose, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, the copolymer-1 of treatment effective dose, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

According to another aspect of the present invention, the method of the agglutination of a kind of skin of inducing or quickening to damage or skin trauma is provided, and described method comprises the expression and/or the activity of regulating the skin or at least a PKC isotype in the skin trauma place hypodermal cell that are settled in damage; Give at least a hormone that is selected from of described hypodermal cell administering therapeutic effective dose, somatomedin, the adipose cell hormone, other reagent of PKC δ RACK and GW9662 and the expression and/or the activity of regulating described PKC isotype, thus induce or the skin that quickens to damage or the agglutination of skin trauma.

According to another aspect of the present invention, the pharmaceutical composition of the agglutination of a kind of skin that is used to induce or quickens to damage or skin trauma is provided, described pharmaceutical composition comprises, as active component, expression or the active material and at least a hormone that is selected from of at least a PKC isotype of adjusting of treatment effective dose, somatomedin, the adipose cell hormone, other reagent of PKC δ RACK and GW9662, and pharmaceutically acceptable carrier.

According to the further feature of the preferred embodiment of following invention, described wound is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, psoriasis wound, saborehic dermatitis wound, the animal skin wound, the animal diabetic wound, retinopathy wound, oral wounds (mucositis), the encolpitis wound, the gingival wound, laceration (laceration), operative incision wound and postoperative intestinal adhesion wound (adhesis wound).

According to the further feature of described preferred embodiment, described ulcer is diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

According to the further feature of described preferred embodiment, described insulin is recombinated.

According to the further feature of described preferred embodiment, described insulin is a natural origin.

According to the further feature of described preferred embodiment, described other reagent is platelet-derived somatomedin.

According to the further feature of described preferred embodiment, described other reagent is the PKC-alpha inhibitor.

According to the further feature of described preferred embodiment, administration is a single administration.

According to the further feature of described preferred embodiment, described outmoded skin trauma is at least 2 days ages (2day sold).

According to the further feature of described preferred embodiment, described insulin concentration is that 0.1 μ M is to 10 μ M.According to the further feature of described preferred embodiment, the dosage unit of insulin be in the pharmaceutical composition of 0.01-0.2ml 0.001 to 5nM.

According to the further feature of described preferred embodiment, the dosage of described insulin be in the pharmaceutical composition of 0.01-0.2ml 0.01 to 0.5nM.

According to the further feature of described preferred embodiment, described pharmaceutical composition is selected from aqueous solution, gel, Emulsion, paste, lotion, spraying, suspension, powder, dispersant, ointment and ointment.

According to the further feature of described preferred embodiment, described pharmaceutical composition contains solid support.

According to the further feature of described preferred embodiment, described cell is transformed to produce and excreting insulin.

According to the further feature of described preferred embodiment, the PDX1 gene transformation of being recombinated of described cell, thus make described cell produce and secrete natural insulin.

According to the further feature of described preferred embodiment, described cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the endogenous insulin gene of described cell, thereby described cell produces and the secretion natural insulin.

According to the further feature of described preferred embodiment, described insulin secretory cell can form secretory granule.

According to the further feature of described preferred embodiment, described insulin secretory cell is endocrine cell.

According to the further feature of described preferred embodiment, described insulin secretory cell is the people source.

According to the further feature of described preferred embodiment, described insulin secretory cell is a histocompatibility peopleization animal origin.

According to the further feature of described preferred embodiment, described insulin secretory cell secretion insulin human.

According to the further feature of described preferred embodiment, described insulin secretory cell is an autogenous cell.

According to the further feature of described preferred embodiment, described cell is selected from fibroblast, epithelial cell and keratinocyte.

According to the further feature of described preferred embodiment, described cell is transformed with the agent of generation Protein kinase C transcriptional activation, thereby makes described cell produce natural Protein kinase C.

According to the further feature of described preferred embodiment, described cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the intrinsic protein kinase c of described cell, thereby makes described cell produce natural Protein kinase C.

According to the further feature of described preferred embodiment, described cell is by the gene transformation of recombiant protein kinase c, thereby makes described cell produce the recombiant protein kinase c.

According to the further feature of described preferred embodiment, described Protein kinase C is selected from PKC-β 1, PKC-β 2, PKC-γ, PKC-θ, PKC-λ and PKC-τ.

According to the further feature of described preferred embodiment, described Protein kinase C is selected from PKC-α, PKC-δ, PKC-ε, PKC-η and PKC-ζ.

According to the further feature of described preferred embodiment, in being suitable for the pharmaceutical composition of local application, contain copolymer-1.

According to the further feature of described preferred embodiment, described PKC isotype is selected from PKC-α, PKC-β, PKC-δ and PKC-ζ.

According to the further feature of described preferred embodiment, described hormone is an insulin.

According to the further feature of described preferred embodiment, described somatomedin is selected from IL-6, KFG and TNF α.

According to the further feature of described preferred embodiment, described adipose cell hormone is adipsin or adiponectin (adiponectin).

The present invention has successfully solved the shortcoming of existing known technology, provides to be used to resist the skin of damage or the new Therapeutic Method of skin trauma.

Description of drawings

Only mode and the appended accompanying drawing of reference are described by way of example in the present invention.Now concrete details with reference to the accompanying drawings should be understood that shown details only is a way of example, to be used for the preferred embodiments of the invention are illustrated, and to be in order providing principle of the present invention and notion is the most useful and the description of easy understanding.From this point, describe except the present invention there being the necessary content of basic comprehension can be again not carry out more detailed constructional details the present invention, can make one of ordinary skill in the art understand how various forms of the present invention is implemented for the description of accompanying drawing.

In the accompanying drawings:

Fig. 1 has proved that the use recombinant adenoviral vector can effectively cross expression PKC isotype: the left side: four day age former generation keratinocyte with β-gal adenovirus infection 1 hour.Infected back 48 hours, fixed cell is by inducing the quantitatively definite proteic activation of beta galactosidase of blue reaction and comparing with the keratinocyte that does not infect.The right: the former generation keratinocyte isotype specificity PKC adenovirus infection 1 hour of recombinating in four day age.Behind the twenty four hours, extract the albumen that infects culture (Ad) and do not infect control cultures (C) and carry out the Westernblot analysis, resist-PKC antibody analysis sample with the described isotype specificity of following embodiment part.

Fig. 2 has shown bryostatin 1 (bryostatin 1) is crossed the PKC isotype of expression to the activation-inducing of PKC migration.The specific reorganization of the former generation keratinocyte usefulness isotype PKC adenovirus infection in four day age 1 hour.Infect the back twenty four hours, cell is not handled (C) or was stimulated classification then 30 minutes with bryostatin 1 (B).Protein sample is carried out Westernblot, resist-the PKC antibody analysis with the isotype specificity.

Fig. 3 has shown that the native form of crossing the PKC isotype of expressing has activity.The former generation keratinocyte usefulness isotype specificity reorganization PKC adenovirus infection in four day age 1 hour.Infected back 18 hours, the control cells (C) of not infection and the cell lysate of PKC isotype overexpressing cell (OE) are carried out immunoprecipitation with the anti-PKC antibody of isotype specificity.Immunoprecipitate is used to carry out the active detection of PKC, as described in following embodiment part.

Fig. 4 has proved that crossing the specificity PKC isotype of expressing induces tangible morphological change in former generation keratinocyte.In former generation,, keratinocyte do not handle (C) or with reorganization PKC α, δ, η, or ζ adenovirus infection.Behind the twenty four hours, with bright field microscope culture is observed and taken pictures (x20).

Fig. 5 has shown and has crossed the PKC isotype of the expressing clear location in infected former generation keratinocyte.With former generation keratinocyte be seeded on the glass slide that laminin 5 (laminin 5) applies.Culture is not handled or with different reorganization PKC adenovirus infections.Infect the back twenty four hours, fixed cell, washing is also air-dry.With FITC bonded two is anti-culture is carried out immunofluorescence analysis then with the anti-PKC antibody of isotype specificity.Scan with the Laser Scanning Confocal Microscope pair cell, taken pictures in the typical visual field.

Fig. 6 has proved the expression of PKC isotype specificity adjusting alpha 6 beta 4 integrin.PKC δ is not handled or used to former generation mouse skin keratinocyte in five day age, PKC α, and PKC η or PKC ζ recombinant adenovirus infect.Infected the back 48 hours, and carried out the SDS-PAGE electrophoresis, go on the nitrocellulose filter, carry out immunoblotting and analyze with ECL with anti-α 6 and anti-β 4 antibody with the theca cell component.

Fig. 7 has shown PKC η that crosses expression and the propagation that PKC δ induces keratinocyte.PKC δ is not handled or used to former generation mouse skin keratinocyte of five days, PKC α, and PKC η or PKC ζ recombinant adenovirus infect.Infected the back 48 hours, carried out 1 hour ³The H-thymidine mixes with analysis of cells propagation, described in the experiment step.The result is expressed as the cpm/ culture dish, compares with the keratinocyte that beta galactosidase infects.Each value representation is the mean+SD of three replication values in 3 independent experiments.

Fig. 8 has proved expressing the localized effect of the hemi desmosome of alpha 6 beta 4 integrin excessively of PKC isotype.In former generation,, keratinocyte was seeded on the glass slide that laminin 5 applies, with the keratinocyte culture at low Ca ²⁺MEM in kept 48 hours.Culture is not handled (A) then, perhaps by PKC α, and PKC δ, PKC η or PKC ζ recombinant adenovirus infect (being respectively B-E).Infect the back twenty four hours, with 4% paraformaldehyde fixed angles keratinocyte cells, extract with 0.2%Triton-X-100 is gentle then, with PBS washing and air-dry, described in the experiment step.With FITC bonded two is anti-culture is carried out immunofluorescence analysis then with anti-α 6 antibody of isotype specificity, described in the experiment step.

Fig. 9 A-B has shown the PKC δ and separate (detachment) of PKC ζ at external evoked keratinocyte that crosses expression.(A) keratinocyte is not handled (C) or by reorganization PKC α, δ, η or ζ adenovirus infection former generation.Infected back 24 hours and 48 hours analysis of cells adhere to, extract cell and once more they are inoculated on the culture dish that substrate applies.Cell number is expressed as the protein concentration (mg/ culture dish) of attached cell.(B) keratinocyte is not handled (C) or by reorganization PKC α, δ, η or ζ adenovirus infection former generation.Infect the floating cell of collecting the disengaging in the culture medium in back 24 hours, the separation of pair cell is analyzed.Cell number is expressed as the protein concentration (mg/ culture dish) of isolated cell.

Figure 10 has proved and has actively expressed PKC η in the keratinocyte of propagation.Former generation keratinocyte inoculation (plate) is on the glass slide that laminin 5 applies.Cultivate keratinocyte and BrdU solution 1 hour inoculation back 48 hours, uses anti-PKC η (redness) and anti-BrdU (green) antibody to carry out immunofluorescence analysis then, and embodiment part described as follows is described.Scan with the Laser Scanning Confocal Microscope pair cell, taken pictures in the typical visual field.

Figure 11 has proved that PKC η induces, and PKC η mutant reduces keratinocyte propagation.In former generation,, the recombinated dominant negative mutant (DNPKC η or PKC DN η) of PKC η adenovirus or PKC η adenovirus of skin keratin cell infected 1 hour.Infected the back 48 hours, carried out 1 hour ³The H-thymidine mixes with analysis of cells propagation, as described in following embodiment part.The result is expressed as cpm/ culture dish.Contrast is the cell that does not infect.

Figure 12 A-B has proved that the mistake of PKC η and DNPKC η expresses location and the cellular morphology of specific regulating PKC.In former generation,, the recombinated dominant negative mutant (PKC DN η) of PKC η adenovirus or PKC η adenovirus of skin keratin cell infected 1 hour.Infected the back 48 hours, fixed angles keratinocyte cells and (A) carry out that bright field is taken a picture (x20) and (B) resist with FITC bonded two then with PKC η specific antibody and carry out immunofluorescence analysis is as testing described in the step.The cell of contrast for not infecting.

Figure 13 A-B has shown that PKC η expresses in the keratinocyte of propagation inhibition induces the differentiation of keratinocyte.In former generation,, the skin keratin cell was at low Ca ²⁺Culture medium in keep propagation or at 0.12mM Ca ²⁺Condition under the differentiation 24 hours.Then, the dominant negative mutant (PKC DN η) with reorganization PKC η adenovirus or PKC η adenovirus infected keratinocyte 1 hour.Infect the back twenty four hours, keratinocyte is remained on low Ca ²⁺Culture medium in or go to and contain 0.12mM Ca ²⁺Division culture medium in kept again 24 hours.Infected the back 48 hours, and extracted keratinocyte and use the SDS-PAGE gel analysis.Analyze the expression of PKC η (A) and keratin 1 (B) with Western blotting.

Figure 14 has proved that in the cutting wound of mice the local expression of PKC η increases the formation and the accelerated wound healing of granulation tissue in the body.Cause the otch of the monoblock skin of 7mm at the nude mice back.1 day and local application in 4 days contrast β-gal, PKC η and PKC α adenovirus suspension after wound takes place.The monoblock skin trauma is fixed in 4% paraformaldehyde, uses H﹠amp; E dyeing and bright field microscope carry out histologic analysis to skin biopsy.E is an epidermis, and D is a corium.

Figure 15 has proved insulin in the keratinocyte of propagation, rather than IGF1 induces the migration of PKC δ specifically.Separate the also inoculation of former generation keratinocyte, as described in following embodiment part.At low Ca ²⁺Culture medium (0.05mM) in keep the keratinocyte 5 days of propagation, reach 80% until them and converge.With cell with 10 ^-7M insulin (Ins) or 10 ^-8M IGF1 (IGF) stimulated 15 minutes.Dissolved cell as mentioned above carries out SDS-PAGE and shifts with 20 μ g films of the contrast that stimulated or do not stimulate (Cont) cell or cytosol extract.Use at the specific polyclonal antibody of each PKC isotype trace is surveyed.

Figure 16 has shown it is the activity that insulin rather than IGF1 can induce PKC δ.In order to determine the activity of PKC δ, with five days keratinocyte cultures with 10 ^-7M insulin (Ins) or 10 ^-8M IGF1 (IGF) stimulates the specified time (1,15, or 30 minutes).With the anti-PKC anti-of specificity from film (blue bar, mem) and cytosol (the purple bar cyto) in the component comes out PKC δ immunoprecipitation.With the PKC activity of vitro kinase check and analysis PKC δ immunoprecipitate, described in the experiment step.Each bar is represented the meansigma methods ± SE of 3 measured values in 3 independent experiments.Each value representation be pmol ATP/ culture dish/minute.

Figure 17 A-B has shown that insulin and IGF1 have for keratinocyte propagation and has added and effect.The keratinocyte of propagation is at low Ca ²⁺Culture medium (0.05mM) in keep reaching 80% until them in 5 days and converge.(A) five days keratinocyte culture stimulated 24 hours with the insulin or the IGF1 of prescribed concentration.(B) simultaneously, with keratinocyte with 10 ^-7The IGF1 (IGF) that M insulin (Ins) and dosage increase stimulates.In each concentration, right bar (striped bar) expression adds observed propagation together with two hormones.Left side bar has proved 10 ^-7The independent effect of IGF1 (grey bar) that M insulin (red bar) and concentration increase.As test and detect mixing of thymidine described in the step.The result has represented six result of experiment.Each bar all is the meansigma methods ± SE of 3 measured values, is expressed as the percent of the keratinocyte that does not stimulate that is higher than contrast.

Figure 18 A-B has proved the expression of crossing of reorganization PKC adenovirus construct.With containing wild type PKC δ (WTPKC δ), wild type PKC α (WTPKC α), or the recombination adenovirus construction body of dominant PKC δ mutant (DNPKC δ) infection keratinocyte culture.(A) infect after with cell culture 24 hours, collect, 20 μ g protein extracts are carried out Western blotting with anti-PKC α of specificity or anti-PKC anti-analyze.The trace result has represented the result of 5 independent experiments.(B) infected the back 24 hours, collecting cell is with vitro kinase check and analysis PKC α or PKC δ immunoprecipitate.

Figure 19 has shown that PKC crosses the effect of expression for insulin or the inductive propagation of IGF1.(light blue) of Gan Raning not, or the cell of crossing expression WTPKC δ (dark blue) or DNPKC δ (oblique line Blue Streak) is with 10 ^-7M insulin (Ins), 10 ^-8M IGF1 (IGF) or the two (Ins+IGF) handled 24 hours.As test and detect mixing of thymidine described in the step.Meansigma methods ± the SE of 3 measured values of 3 experiments that each bar is represented to carry out with independent culture.Each value representation is the percent based on the keratinocyte that does not stimulate of same culture in each experiment in contrast.

Figure 20 has shown the propagation of the keratinocyte that the active inhibition specificity elimination of PKC δ is insulin-induced.As cultivation keratinocyte of former generation as described in the following embodiment part.The keratinocyte that infects of cell of Gan Raning or DNPKC δ was not with the factors stimulated growth of following concentration 24 hours: 10 ^-7M insulin (Ins), 10 ^-8M IGF1 (IGF), 10ng/ml EGF, 10ng/ml PDGF, 1ng/ml KGF or 5ng/ml ECGF.As mixing of detection thymidine as described in the following embodiment part.Meansigma methods ± the SE of 3 measured values of 3 experiments that each bar is all represented to carry out with independent culture.Each value representation is the percent based on the keratinocyte that does not stimulate of same culture in each experiment in contrast.

Figure 21 has shown that crossing the insulin-induced keratinocyte of PKC δ specific regulating of expressing breeds.In former generation,, keratinocyte cultivated as described in Figure 1 like that.Cell of Gan Raning or the keratinocyte that infects with the WTPKC δ that cross to express be not with the factors stimulated growth of following concentration 24 hours: 10 ^-7M insulin (Ins), 10 ^-8M IGF1 (IGF), 10ng/ml EGF, 10ng/ml PDGF, 1ng/ml KGF or 5ng/ml ECGF.As mixing of detection thymidine as described in the following embodiment part.Meansigma methods ± the SE of 3 measured values of 3 experiments that each bar is all represented to carry out with independent culture.Each value representation is the percent based on the keratinocyte that does not stimulate of same culture in each experiment in contrast.

Figure 22 A-B has confirmed that PKC δ and PKC ζ have important function in the skin wound healing process in vivo.Use the interior mouse model of body of the invalid mice of isotype specificity PKC newly developed, to PKC α, the brood birth mice of PKC δ and the invalid mice of PKC ζ and their wild type carries out wound healing research.Anesthetized mice also causes the skin of diameter 4mm by aspiration biopsy at the mice back.Take off mouse skin after the tracking of carrying out a week, the chamber method is carried out the wound strength test to determine the healing of skin trauma to the skin thin slice with breaking.Its value is expressed as fracture pressure, and its representative is indoor until disruptive maximum pressure takes place in monitoring.The result is not on the same group the measured value of 12-20 mice.Experiment repeats 3 times at least.

Figure 23 has determined the special interaction between the STAT3 and PKC δ in former generation skin keratin cell.Former generation keratinocyte do not handle (on) or with isotype specificity reorganization PKC adenovirus infection 1 hour (descending).Extract cell and carry out immunoprecipitation (IP) with isotype specificity PKC antibody.With anti-PKC or anti-STAT3 antibody immunoprecipitate being carried out Western blot analyzes.

The activation that Figure 24 has proved PKC δ has important function for the transcription activating of insulin-induced STAT 3.With former generation keratinocyte be seeded on the glass slide, at low Ca ⁺⁺Keeping reaching 80% until them in 5 days in the culture medium (0.05mmol/l) converges.Cell does not handle (Cont, on) or (R down), used 10 then with 5 μ M Rottlerin pretreatment 7 minutes ^-7The M insulin is handled 5 minutes (Ins).Use the methanol fixed cell, washing is also air-dry.Use FITC bonded two anti-immunofluorescences to analyze culture then with anti-phosphoric acid-Tyr-705-STAT3 antibody.Use the scanning cofocal microscope cell.

Figure 25 has proved to cross and has expressed the propagation that DN PKC δ suppresses the keratinocyte of crossing induced expression of PKC δ and STAT3.In former generation,, keratinocyte was with containing β-Gal (in contrast), PKC δ, and WT STAT3, the recombination adenovirus construction body of DN STAT3 infected 1 hour or with DN PKC δ, carried out double infection with STAT3 then.Infected back 24 hours, carried out 1 hour ³The H-thymidine mixes with analysis of cells breeds.The result is expressed as DPM/mg protein.Each bar all is the meansigma methods that the flat board (plate) with same culture is measured carry out for three times.

Figure 26 has proved the effect for wound healing in the body of the concentration of insulin administration and frequency.C57BL mouse back in age in 8-10 week cuts out the wound otch, carries out insulin processing (in just seven days every day repetitive administration is with respect to using once separately) with PBS (contrast) processing or with variable concentrations and frequency.Cause and create back seven days, detect the wound site of handling mice execution.The result is expressed as mm ²Wound site, each bar all are six multiple mean+SD (p＜0.005).

Figure 27 has proved the concentration of insulin administration and the frequency histology's effect for wound healing in the body.C 57BL mouse back in age in 8-10 week cuts out the wound otch, carries out insulin with variable concentrations and frequency and handles (in just seven days every day repetitive administration is with respect to using once separately).Cause the section of wound preparation in back seven days histology wound, analyze the closure (wound contraction) of epidermis and corium.The closure of epidermis detects with Keratin 14 (K14) antibody staining (left side), if the whole breach of wound all is a stained positive, then is considered as the positive.If (right side) can both seen under the amplification of x100 in the both sides of corium wound in a visual field under optical microscope, then the closure of corium is considered as the positive.The result is expressed as the percent of wound closure with respect to contrast, and each bar all is six multiple meansigma methodss.

Figure 28 has proved that (platelet derivedgrowth factor PDGF-BB) is used in combination synergism for wound healing in the body for insulin and platelet derived growth factor.C57BL mouse back in age in 8-10 week cuts out the wound otch, uses insulin, PDGF-BB, or be used in combination insulin and PDGF-BB carries out single administration.Cause wound and put to death mice in back seven days, get the histologic analysis that living tissue is carried out epidermis and corium closure (wound contraction).The closure of epidermis detects with Keratin 14 (K14) antibody staining (left side), if the whole breach of wound all is a stained positive, then is considered as the positive.If (right side) can both seen under the amplification of x100 in the both sides of corium wound in a visual field under optical microscope, then the closure of corium is considered as the positive.The result is expressed as bar diagram, is the percent of wound closure with respect to contrast, and each bar all is six multiple meansigma methodss.

Figure 29 A-D illustrates insulin and PKC alpha inhibitor to be used in combination photo to the morphology effect of wound healing in the body.C57BL mouse back in age in 8-10 week cuts out the wound otch, does not handle (contrast) or with insulin (HO/01) and PKC alpha inhibitor (HO/02) combined treatment.Causing wound removed the skin living tissue to carry out morphological observation in back 7 days.Figure 29 A-B has shown the contrast wound, and Figure 29 C-D has shown the wound of handling.

Figure 30 illustrates insulin and PKC alpha inhibitor to be used in combination the microphotograph of organizing to the effect of corium closure (wound contraction).C57BL mouse back in age in 8-10 week cuts out the wound otch, does not handle (contrast) or with processing every day that combines of insulin (HO/01) and PKC alpha inhibitor (HO/02).Cause wound and put to death the mice of handling in back seven days.Making histology wound section is also observed under optical microscope.If the both sides of corium wound can both see that then the closure of corium is considered as the positive under the amplification of x100 in a visual field.The open wound area of untreated contrast section (left side) can not be contained in too greatly in the visual field that an x100 amplifies, and that the wound of handling section (right side) shows corium is closed positive.The xanthelasma dotted line has marked the edge of corium.

Figure 31 illustrates insulin and PKC alpha inhibitor to be used in combination the microphotograph of organizing to the effect of epidermis closure.C57BL mouse back in age in 8-10 week cuts out the wound otch, does not handle or with processing every day that combines of insulin (HO/01) and PKC alpha inhibitor (HO/02).Cause wound and put to death the mice of handling in back seven days.The tissue slice of preparation wound with keratin 14 (indication substrate keratinocyte) dyeing, is observed under optical microscope.Open wound area (arrow marks) in the untreated contrast section (left side) can not be contained in too greatly in the visual field of an x100 amplification, and the wound of handling section (right side) whole breach shows the epidermis closure.

Figure 32 illustrates insulin and PKC alpha inhibitor to be used in combination the microphotograph of organizing to the effect of epidermis cell space differentiation.Causing wound mice (C57BL, 8-10 week age) handles with local application every day that combines of insulin (HO/01) and PKC alpha inhibitor (HO/02).Cause wound and put to death the mice of handling in back seven days.The tissue slice of preparation wound is with keratin 1 (K1) antibody staining of the starting stage that highlights the cell spaces differentiation.Untreated contrast section (left side) shows big undifferentiated wound area (being marked by arrow), then observes a large amount of epidermal renewals in the wound section of handling (right side).

Figure 33 has proved that insulin and PKC alpha inhibitor are used in combination the quantitative effect to wound healing in the body.Causing wound mice (C57BL, 8-10 week age) handles with local application every day that combines of insulin (HO/01) and PKC alpha inhibitor (HO/02).Cause wound and put to death the mice of handling in back seven days.The tissue slice of preparation wound carries out corium and shrinks, and the analysis that epidermis is closed and the space breaks up is as described in above-mentioned Figure 30-32.Bar diagram has demonstrated the incidence rate (percent) of the wound that heals fully in each processed group that draws by histologic analysis.

Figure 34 A-G is the expression of PKC α in the hypodermal cell and/or the expression and/or the active adjusting of active inhibition and another kind of PKC isotype, perhaps uses the example photo of hormone for the combined effect of the closure of vitro skin wound to hypodermal cell.Former generation skin flbroblast of cultivating is infected with the PKC α of the kinases inactivation of dominant (DN).Implement scraping behind the twenty four hours, culture is not handled (Figure 34 A), perhaps uses wild type (WT) PKC δ (Figure 34 B), PKC η (Figure 34 C), and WT PKC ζ (Figure 34 D) or WT PKC ε (Figure 34 E) infect.In addition, culture adipsin (the 2 μ g/ml of PKC α inhibition; Figure 34 F) or insulin (6.7 * 10 ^-7M; Figure 34 G) handles.Handle and took pictures in back 24 hours.

Figure 35 A-H is the expression of PKC α in the hypodermal cell and/or the expression and/or the active adjusting of active inhibition and another kind of PKC isotype, perhaps uses the example photo of somatomedin for the combined effect of the closure of vitro skin wound to hypodermal cell.Former generation skin flbroblast of cultivating is infected with the PKC α of the kinases inactivation of dominant (DN).Implement scraping behind the twenty four hours, culture is not handled (Figure 35 A), perhaps uses wild type (WT) PKC ε (Figure 35 D), and WT PKC ζ (Figure 35 E) or WT PKC η (Figure 35 F) infect.Perhaps, culture IL-6 (each culture dish 1 μ g of PKC α inhibition; Figure 35 B), KGF (each culture dish 1 μ g; Figure 35 C), PKC δ RACK (10 ^-7M; Figure 35 H) or TNF α (12 μ g/ml; Figure 35 G) handles.Handle and took pictures in back 24 hours.

Figure 36 A-B is the expression of PKC ζ in the hypodermal cell and/or active inhibition and uses the example photo of somatomedin for the combined effect of the closure of vitro skin wound to hypodermal cell.Former generation skin flbroblast of cultivating is infected with the PKC ζ (DN ζ) of the kinases inactivation of dominant (DN).Implement scraping behind the twenty four hours, culture is not handled (Figure 36 A), perhaps uses KGF (each culture dish 1 μ g; Figure 36 B) handles.Handle and took pictures in back 24 hours.

Figure 37 A-D is the expression of PKC ζ in the hypodermal cell and/or active inhibition and uses somatomedin or the hormone example photo for the combined effect of the closure of vitro skin wound to hypodermal cell.Former generation skin keratin cell of cultivating is infected with the PKC ζ (DN ζ) of the kinases inactivation of dominant (DN).Implement scraping behind the twenty four hours, culture is not handled (Figure 37 A), perhaps uses IL-6 (each culture dish 1 μ g; Figure 37 B), TNF α (12 μ g/ml; Figure 37 C) or adiponectin (each culture dish 1 μ g; Figure 37 D) handles.Handle and took pictures in back 24 hours.

Figure 38 A-E is the expression of PKC β in the hypodermal cell and/or active inhibition and uses somatomedin to hypodermal cell that insulin or GW9662 are for the example photo of the combined effect of the closure of vitro skin wound.Former generation skin flbroblast of cultivating is infected with the PKC β (DN β) of the kinases inactivation form of dominant (DN).Implement scraping behind the twenty four hours, culture is not handled (Figure 38 A), and (each cultivates 1 μ g perhaps to use KFG; Figure 38 B), (each cultivates 1 μ g to IL-6; Figure 38 C), insulin (6.7 * 10 ^-7M; Figure 38 D) or GW9662 (each culture dish 1 μ g; Figure 38 E) handles.Handle and took pictures in back 24 hours.

Figure 39 A-E is the expression of PKC δ in the hypodermal cell and/or the expression and/or the active adjusting of active inhibition and another kind of PKC isotype, perhaps uses the example photo of hormone for the combined effect of the closure of vitro skin wound to hypodermal cell.Former generation skin keratin cell of cultivating is infected with the PKC δ (DN δ) of the kinases form of wild type (WT).Implement scraping behind the twenty four hours, culture is not handled (Figure 39 A), perhaps uses WT PKC ζ (PKC ζ; Figure 39 B), WT PKC ε (PKC ε; Figure 39 C) or DN PKC α (PKC α; Figure 39 D) infects.Perhaps, culture adipsin (the 2 μ g/ml of PKC δ raising; Figure 39 E) handles.Handle and took pictures in back 48 hours.

Figure 40 A-F uses copolymer-1, insulin, and the pseudo-substrate of PKC α, or its combination is for the example photo of the effect of vitro skin wound closure.Former generation skin keratin cell of cultivating is not handled (Figure 40 A), perhaps only uses insulin (6.7 * 10 ^-7M; Figure 40 B), only use copolymer-1 (55 μ g/ culture dishs; Figure 40 C), the mixture of the pseudo-substrate of insulin and PKC α (is respectively 6.7 * 10 ^-7M and 10 ⁷M; Fig. 4 OD), the mixture of copolymer-1 and insulin (is respectively 55 μ g/ culture dishs and 6.7 * 10 ^-7M; Figure 40 E), or copolymer-1, the mixture of the pseudo-substrate of insulin and PKC α (is respectively 55 μ g/ culture dishs, 6.7 * 10 ^-7M and 10 ⁷M; Figure 40 F) handles.Handle and took pictures in back 48 hours.

Figure 41 A-D is a copolymer-1, insulin, and the pseudo-substrate of PKC α, or its combination is for the example photo of the effect of wound healing in the body.Cause the wound mice and do not handle (Figure 41 A) or local application every day copolymer-1 (55 μ g/ml in 4 days; Figure 41 B), the mixture of copolymer-1 and insulin (is respectively 55 μ g/ml and 1 μ M; Figure 41 C), or copolymer-1, the mixture of the pseudo-substrate of insulin and PKC α (is respectively 55 μ g/ml, 1 μ M and 1 μ M; Figure 41 D).Cause wound took pictures in back 4 days.

Figure 42 A-H is near the thymus of the wound breach microphotograph of organizing for the example of the effect of wound healing process.Figure 42 A-B has shown 200 times of (x200) enlarged drawings of the normal rodentine thymus of growing up.Figure 42 C has shown 40 times of (x40) enlarged drawings of wound in 7 day age, is observing thymus (red square is amplified 200 times, Figure 42 D) from the very approaching place of wound breach.Wound forms epithelium again, forms granulation tissue, and corium shrinks.Figure 42 E-F has shown the wound in 9 day age of amplifying the STZ diabetic mice of 40 times (Figure 42 E) and 200 times (Figure 42 F), does not observe thymus near the wound breach, do not observe and form epithelium again, and granulation tissue, or corium shrinks.Figure 42 G has shown the wound in 9 day age of the STZ diabetic mice that amplifies 40 times.With the mixture process of wound with insulin and the pseudo-substrate of PKC α.Near the wound breach, observe thymus (red square is amplified 20 times, Figure 42 H).Wound forms epithelium again, forms granulation tissue, and corium shrinks.

Figure 43 is that insulin and PKC alpha inhibitor are used in combination the example photo to the effect of wound and injured skin healing.At Large Whites﹠amp; The back that Landrace family raises pigs forms vertical wound otch, and every day is with the mixture process of PBS (contrast) or 1 μ M insulin and 1 μ M PKC α puppet substrate (HO/03/03) in 15 days.Cause wound took pictures to wound in back 30 days.Compare with the buffer contrast, the wound that HO/03/03 handles heals fully, does not have cicatrization, has significantly increased the aesthetic feeling of skin.

The specific embodiment

The present invention is used to regulate serine/threonine protein kitase, is also referred to as expression and/or the activation of PKC, inducing and/or to quicken cell proliferation and/or cell differentiation, thus the method for the agglutination of acceleration of wound and pharmaceutical composition.According to instruction of the present invention, the adjusting of this expression can realize by following effects: (i) transform the wound cell with the PKC expression construct; (ii) transform the wound cell with cis acting element, this element will be inserted into the endogenous PKC upstream region of gene of wound cell close position; (iii) administration of insulin and other can with the synergistic reagent of insulin to regulate expression and/or the activation of PKC in the wound cell; (iv) transform the wound cell with the insulin expression construct, the expression and/or activatory the going up that can be used as PKC with excretory insulin of the expression of its generation are adjusted; (v) transform the wound cell with cis acting element, this element will be inserted into the endogenous insulin gene of wound cell upstream close position, and the expression and/or activatory the going up that can be used as PKC with excretory insulin of the expression of its generation are adjusted; (vi) in wound, implant insulin secretory cell; (vii) use trans acting factor, for example PDX1 transforms the wound cell, and with the generation and the secretion of inducing endogenous insulin, insulin can be used as expression and/or activatory the going up of PKC and adjusts; And (viii) use the PKC regulator to wound.

With reference to the accompanying drawings and description can understand the principle and the operation of the method for the invention and pharmaceutical composition better.

Before describing at least one embodiment of the present invention in detail, be to be understood that to the invention is not restricted in the following description or illustrative concrete component composition of embodiment part or arrangement.The present invention can be with other embodiment or is implemented in a different manner.And, be to be understood that the phraseology and terminology used herein only are in order to describe use, and should not to be considered as be restriction.

Adult's skin comprises two-layer: the skin corium connective tissue that is rich in collagen of cornified stratified epidermal area and thick-layer below provides support and nutrition.Skin is resisted the external world as the protection barrier.Therefore the damage of any skin or break all must fast and effeciently be repaired.As described in above-mentioned background technology part, first stage of reparation is to block initial wound by the formation blood clotting to realize.Inflammatory cell then, fibroblast and blood capillary are invaded blood clotting and are formed granulation tissue.Follow-up phase comprises that the epithelium of wound rebuilds, and wherein the substrate keratinocyte must lose and being connected of hemi desmosome, and keratinocyte is moved on the granulation tissue, covers wound.After the keratinocyte migration, keratinocyte quickens propagation, and it allows the cell that replacement is lost in damage.After wound is covered by the keratinocyte of monolayer, form new stratified epidermis, rebuild new basement membrane (20-23).Proved that several somatomedin participate in this process, comprised the EGF family of somatomedin, KGF, PDGF and TGF β 1 (22-24).In these somatomedin, the propagation of EGF and KGF and epidermal keratinocytes and the adjusting of migration closely related (25,26).It is important to understand the signal that triggers cell migration, propagation and the substrate that lay is new on the wound breach in the wound for understanding for wound healing biology.

For the ease of understanding following public invention, as a plurality of terms of following definition.

Term " wound (wound) " general reference is any one skin that causes and the hypodermic damage in (for example the decubital ulcer of lying up for a long time and causing, the wound that wound causes, otch, ulcer, burn etc.) in several ways, and its feature has nothing in common with each other.Wound typically can be divided into a kind of in four grades according to the degree of depth of wound: (i) I level: wound is only limited to epithelium; (ii) II level: wound extends to corium; (iii) III level: wound extends to subcutaneous tissue; (iv) IV level (or through thickness wound): the wound (for example outstanding for example bigger trochanter or the rumpbone of pressure spot (bony pressure point) of bone) that exposes bone.

Term " segment thickness wound " refers to comprise the wound of I-III level; The example of segment thickness wound comprises burn, decubital ulcer, venous stasis ulcer, and diabetic ulcer.

Term " depth wound " comprises III level and IV level wound.

Term wound " healing " refers to for example repair the process of wound by forming cicatrix.

Phrase " is induced or is quickened the agglutination of skin trauma " and refers to that the granulation tissue of inducing wound to shrink forms and/or induce epithelium formation (just generating new cell at epithelium).Wound healing can detect by the wound area that reduces easily.

The present invention has considered to treat all injury types, comprises depth wound and chronic trauma.

Term " chronic trauma " refers to not have the wound of healing in 30 days.

Phrase " transformant " refers to that described exogenous nucleic acid is incorporated in the cellular genome by introducing the nucleic acid that exogenous nucleic acid temporarily or for good and all changes cell, changes described cell from heredity, perhaps keeps not integrated state.

Term used herein " cis acting element " is meant gene region as the protein-bonded attachment site of DNA (for example enhancer, operon (operator) and promoter), thereby influences the activity of one or more genes on the same chromosome.

Phrase used herein " trans acting factor " is meant the active factor that combines and regulate its related gene expression with cis acting element.For example, PDX1 combines and regulates its active trans acting factor with the insulin gene promoter.

Phrase used herein " transcriptional activator " is meant the factor that can increase gene expression.Trans acting factor is the example of direct transcriptional activator.

Term used herein " activator " is meant the molecule of enhanced activity.

Phrase used herein " expression that is conditioned and/or activation " is meant expression and/or activation enhanced or that suppress.

PKC is a main signal transduction path, and it can regulate the propagation and the differentiation of keratinocyte.PKC isotype α, δ, ε, η and ζ express (4,10) in skin.When design was of the present invention, imagination PKC expressed and/or activatory adjusting can be bred and/or cell differentiation by inducing cell, thus the agglutination of acceleration of wound.When enforcement is of the present invention, experimental results demonstrate this theory, shown that PKC expresses and/or the agglutination of the certain inducing cell propagation of activatory adjusting and cell differentiation and acceleration of wound.In further specifically describing, adopted multiple diverse ways to regulate expression and/or the activation of PKC, thus the agglutination that has quickened wound.Find based on these experiments, designed other method.Found that when enforcement is of the present invention a noticeable new phenomenon-insulin can be used as PKC and expresses and/or activatory regulator.Therefore, insulin can be used as regulate that PKC expresses and/or activatory therapeutic agent with the agglutination of acceleration of wound.

The feature of different PKC isotypes and they are very important for the effect of cell proliferation and/or differentiation for skin wound healing biology.Use the PKC adenovirus construct can differentiate of the specific function of various PKC isotypes for wound healing process in external and the body.All isotypes can both specifically influence the different aspect of keratinocyte growth and differentiation.Two isotypes, PKC δ and PKC ζ can specifically be regulated the regulation and control (referring to following embodiment 6) of integrin, with the formation of adhering to (referring to following embodiment 9) and hemi desmosome (referring to following embodiment 8) of basement membrane.Find two kinds of isotypes, PKC δ and PKC η regulate the multiplication potentiality (referring to following embodiment 7 and 11) of epidermal keratinocytes.In addition, the dominant isotype of PKC η (DNPKC η) can be induced differentiation (referring to following embodiment 12) especially in the keratinocyte of actively breeding.At last, also confirmed of the effect of different PKC isotypes in the system in vivo for the skin wound healing process.Use the invalid mice of the ruined PKC of expression of wherein different PKC isotypes, this paper shown the skin keratin cell adhere to necessary PKC δ of transition process and PKC ζ also be very important for wound healing process in the body in the animal model.(referring to embodiment 19).The through thickness biopsy of the whole skin of the skin that PKC is invalid shows that PKC δ and PKC ζ rather than PKC α are essential for the correct healing of wound.And following embodiment 22 has shown that the PKC alpha inhibitor effectively promotes wound healing in the body, thereby shows that PKC α isotype may have antagonism to wound healing.

PKC η has unique tissue distribution.It mainly expresses (27,28) in epithelial tissue.In situ hybridization research and immunohistochemistry studies have shown that PKC η expresses (27) that breaking up with (differentiating and differentiative) differentiation layer camber.The result of this paper shows the functional regulator that can be used as skin proliferation and differentiation according to cytophysiology PKC η.When keratinocyte at low Ca ²⁺When maintaining vegetative state under the condition, the speed that PKC η induces propagation is five to seven times of the contrast keratinocyte.But, when passing through to increase Ca ²⁺In the time of the differentiation of concentration inducing cell, compare differentiation with control cells and induced (referring to embodiment 12) with faster and higher speed.This can explain the ability that PKC η significantly induces wound healing and granulation tissue to form, because multiplication capacity and differentiation layer form has all realized.What is interesting is, wound healing result and the expression of PKC η in embryonal tissue in the body, its usually the manhood not with high level expression PKC η, show that PKC η may have effect in the propagation of other tissue and organizational structure.This comprises nerve and corium and muscular tissue, and they can effectively healing in the granulation tissue of wound.And, actively regulating the differentiation of keratinocyte especially and inducing the ability of normal differentiation to make the hyper-proliferative that can handle especially in differentiation and the control wound healing process lack of proper care in the proliferating cells by using dominant (dominantnegative) mutant.

This paper understands that for example the healing ability of PKC η is to apply in the body on the wound that produces at the nude mice back.Following embodiment 14 shows after local infection four days, and wound is used the formation that PKC η expression construct causes granulation tissue.

Generally speaking, the result of this paper has proved that expression and/or the activation (film migration) of regulating different PKC isotypes are the effective tools of antagonism wound.Correspondingly, can be by increasing isotype PKC δ, expression and/or the activity of PKC η and PKC ζ perhaps promote wound healing by expression and/or the activity that suppresses isotype PKC α.

Therefore, according to an aspect of the present invention, provide a kind of method of inducing or quickening the agglutination of skin trauma or damage, described method expresses by at least a adjusting PKC that gives skin trauma administering therapeutic effective dose and/or activatory reagent is realized.According to this aspect of the present invention, therefore the pharmaceutical composition that is used to implement this method comprises that being used to regulate PKC as the treatment effective dose of active component at least a expresses and/or activatory reagent; And pharmaceutically acceptable carrier.

Phrase used herein " skin trauma " is meant the epithelium wound of any kind, includes but not limited to ulcer, for example diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HI V related ulcers on, the wound that diabetes are relevant, burn, sunburn, the aging skin wound, the corneal ulcer wound, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound (adhesis wound).

Phrase used herein " skin injury " for example is meant the skin injury of any kind or situation, wrinkle (for example ultraviolet radiation cause wrinkle), skin stricture of vagina, the crack, lump, big pore is (for example with accessory structure such as sweat duct, sebaceous gland, or hair follicle is relevant), uneven or coarse, skin follows the string (functional skin elasticity proteins lose and/or inactivation), sagging (comprising eye and lower jaw edema), skin hardness (firmness) forfeiture, the forfeiture of the skin degree of packing, the recovery capacity forfeiture after the skin deformation, variable color (comprising black eye), the speckle bleb, colour of skin sallow, pigment excessive skin zone is senile plaque and freckle for example, horn, unusual differentiation, excessive keratinization, elastosis, the destruction of collagen protein, and skin keratin, corium, epidermal area, skin heart system (for example telangiectasis or pitch blood vessel) more, and subcutaneous tissue, particularly change near other tissue in the subcutaneous tissue of skin.

Skin is not considered to typical insulin response sex organization.Therefore the effect of insulin in skin mainly belongs to the ability that it activates the IGFR that is closely related.Shown that in keratinocyte insulin and IGF1 can costimulatory receptors, activate similar downstream effect (34).But, though having proved these two somatomedin, the present invention can both induce keratinocyte propagation in dose-dependent mode, every kind of hormone all is to play a role with different signal transduction paths.The insulin of pointing out at first and IGF1 are confirmed by following discovery the different adjusting of keratinocyte propagation: breed for keratinocyte when these hormones add together with the maximum proliferation-inducing concentration of every kind of hormone and have addition (referring to embodiment 15).In order to differentiate insulin and the IGF1 signal transduction path difference in the regulation and control of keratinocyte propagation, detected the known element that can regulate keratinocyte propagation and conduct downstream effect as insulin signaling.These studies show that insulin signaling conducts the adjusting (referring to embodiment 17) that is subjected to PKC δ especially in the propagation of keratinocyte.PKC δ is a kind of isotype of uniqueness in the PKC protein family, participates in the growth and the maturation (35) of various cell types especially.But, though PKC δ is proved to be and is subjected to several somatomedin especially and comprises EGF, the adjusting of the stimulation of platelet derived growth factor and neurotransmitter, but its physiological role has shown it is to have participated in the cell growth to comprise the somatomedin inhibition (36-41) that apoptosis, differentiation and cell cycle postpone or stop.Recently show in the mice keratinocyte of cultivating at rising Ca ²⁺In 12-24 hour, the selectivity of alpha 6 beta 4 integrin complex is lost induce interrelated (6) with K1 after the concentration.The forfeiture of alpha 6 beta 4 protein expression is to transcribe and translate the back incident, comprises the increase of the processing of α 6 and β 4 chains, causes.In preliminary research, set up contact in the processing of the activation of PKC and alpha 6 beta 4 integrin with between regulating.The former result of the effect that forfeiture that these results and relevant PKC δ and PKC ζ express for alpha 6 beta 4 and the isolating hemi desmosome of inducing keratinocyte form is consistent.Yet the present invention has found another effect of PKC δ, is exactly the target as insulin-induced keratinocyte propagation.Following embodiment shows the stimulation of having only insulin, rather than multiple somatomedin, includes but not limited to EGF, KGF, and PDGF, ECGF and IGF1 can make PKC δ, rather than any other PKC isotype of expressing in the skin moves and make its activation.Work as EGF, KGF, PDGF, the mitogenesis of ECGF and IGF1 stimulates when not blocked by the dominant negative mutant of PKC δ, PKC δ further is confirmed to the importance of insulin stimulating, the main activator (referring to embodiment 17) of seemingly this PKC isotype of insulin in the adjusting of keratinocyte propagation.But when keratinocyte was infected by WT PKC δ keratinocyte, the mitogenesis of EGF and KGF stimulates to be increased.This activation that shows PKC δ also is essential for by the stream signal pathway proliferative of other somatomedin being stimulated.And, identified downstream components, it plays regulating action in insulin-induced PKC δ activation and keratinocyte propagation, and has identified STAT3, a kind of transcriptional activator that participates in this process.STAT (signal conduction and transcription activator) albumen is a transcription factor family, comprises the various kinds of cell factor and somatomedin.In seven known STAT family members, STAT3 is unique.Target destroys STAT3 surely rather than other STAT family member causes body early embryo to cause death.Especially, when removing STAT3 conditionally in skin when, the reconstruction of skin is by heavy damage.In case be activated, stat protein forms homodimer or heterodimer, moves in the nucleus, combines with inducible transcription with the DNA response element of target gene.Discovery in keratinocyte, other PKC isotype of expressing in PKC δ rather than the skin (PKC α, ζ, η and ε) relevant with the STAT3 composing type (referring to embodiment 18).And insulin activates the phosphorylation of regulating STAT 3 by the specificity to PKC δ, activation and nuclear migration.By the medicine inhibitor, kamaline or cross is expressed the active inhibition to PKC δ that dominant PKC δ mutant causes can block insulin-induced STAT3 activation and nuclear displacement.Finally, crossing of dominant PKC δ mutant expressed the keratinocyte propagation (referring to embodiment 18) of crossing induced expression that can suppress STAT3.These results show the effect of PKC δ activity insulin-induced in the skin keratin cell proliferation for the transcriptional activation of STAT 3.Because it is extremely important that STAT3 rebuilds for skin, is downstream effect of the various kinds of cell factor and somatomedin, all these results show that the activation of PKC δ is the main downstream components by various skin somatomedin mediation keratinocyte propagation.Especially, PKC δ is the pathogenetic main material standed for of the defective wound healing of diabetic.Contact between PKC δ and the wound healing has also obtained conclusive evidence in vivo.Use the new invalid mice of PKC δ that makes up, this paper has shown the wound healing (referring to embodiment 19) that lacks PKC δ and can postpone mouse skin.In several other systems, also determined the contact between the conduction of PKC δ and insulin signaling.For example, show recently in the muscle culture that PKC δ regulates insulin-induced glucose transport (42,43).Similarly, in the cell of crossing the expression of insulin receptor, show insulin stimulating relevant with the activation of PKC δ (44-46).But though the activation of the PKC δ of insulin-mediated is relevant with the metabolic effects of insulin in these researchs, this is first report that cell proliferation of PKC δ and insulin-mediated is connected.Differentiated that PKC δ plays dual parts in the propagation of keratinocyte and the control of early stage differential period, wherein in the described early stage differential period, cell lost and following basement membrane between adhere to.This shows the main candidate of insulin-induced PKC δ as the physiological equilibrium between propagation of regulating skin and the differentiation.

Therefore, according to instruction of the present invention, express and/or activate by making wound cells contacting islets of langerhans usually regulate PKC.This can be accomplished in several ways, further illustration described as follows.

A kind of mode is directly to give wound with insulin.As described in following embodiment 21 and 22, wound local application insulin is effectively promoted epidermis and corium is closed and subsequently wound healing with the concentration of 0.1-10 μ M.Yet surprising and unexpectedly, insulin and PDGF-BB somatomedin have perhaps produced significant and synergitic promotion than independent administration of insulin to wound healing process with PKC alpha inhibitor combined administration.

Therefore, according to another aspect of the present invention, provide a kind of method of inducing or quickening the agglutination of skin trauma or damage.Described method insulin and at least a and synergistic other reagent of insulin by giving skin trauma administering therapeutic effective dose is realized, to induce or to quicken the agglutination of skin trauma or damage.Preferably, described reagent is the PKC alpha inhibitor.Further preferably, described reagent is for example PDGF of somatomedin, EGF, and TGF β, KGF, ECGF or IGF1, most preferably described reagent is PDGF-BB.

The direct administration of insulin, perhaps individually dosed or with another kind of agent combination administration, can realize by single or repeated application.Implementing the time of the present invention, the inventor surprisingly find concentration single administration insulin with 1 μ M for wound healing than repeat administration every day significantly more effective (referring to following embodiment 20) that carries out seven times with similar concentration.

Therefore, according to another aspect of the present invention, provide a kind of method of inducing or quickening the agglutination of skin trauma or damage, comprised the insulin of using the treatment effective dose of single dose unit to skin trauma.Preferred described single dose unit comprises 0.001 to 5nM, and preferred 0.01 to the 0.5nM insulin, for example, and aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersant, ointment or ointment preparation present in an amount at least sufficient to cover the skin trauma of 1cm area, for example 0.01-0.2ml.

May be very important with the arrangement of time of insulin administration on wound, described in the embodiment 20 in the following embodiment part.For example 4 days wound single administration insulin is produced effective wound healing.Therefore, according to another aspect of the present invention, provide a kind of method of inducing or quickening the agglutination of outmoded skin trauma, comprised the insulin of using the treatment effective dose of single dose to skin trauma.

Phrase used herein " outmoded skin trauma " is meant at least one day, at least two days, at least three days, preferred at least four days skin trauma.

According to another aspect of the present invention, the pharmaceutical composition that is used to induce or quicken the agglutination of skin trauma or damage comprises, as active component, the insulin of treatment effective dose, at least a and synergistic other reagent of insulin, and the pharmaceutically acceptable carrier that is designed for the topical application of pharmaceutical composition.Preferably, described reagent is for example PDGF of PKC alpha inhibitor or somatomedin, EGF, and TGF β, KGF, ECGF or IGF1 most preferably are PDGF-BB.Pharmaceutically acceptable carrier can be but be not limited to gel, cream (cream), and paste, lotion, spraying, suspension, powder, dispersant, ointment (salve) and ointment hereinafter will describe in further detail.Can also use solid support so that wound is prolonged uelralante.Being to be understood that described insulin can be natural or preferably reorganization, can be the people source or any other suitable source.

According to another aspect of the present invention, the pharmaceutical composition that is used to induce or quicken the agglutination of skin trauma or damage can comprise the insulin of the single dose unit of the selected agglutination that can induce or quicken skin trauma or damage, and the pharmaceutically acceptable carrier that is designed for the local application of pharmaceutical composition.Preferably, the insulin of described single dose unit be in the dosage unit of 0.01-0.2ml preparation 0.001 to 5nM, preferred 0.01 to 0.5nM.

In another embodiment of the invention, the cell of expression and excreting insulin is implanted wound, to induce or to quicken the agglutination of skin trauma or damage.This cell that can produce insulin can be the cell of natural generation insulin, perhaps can be transformed the cell with generation and excreting insulin.Described cell can (referring to GeneBankAccession No.AH005712, AF035260 AF035259) transforms, and this gene is the trans acting factor of generation and excreting insulin with the PDX1 gene of for example recombinating.Perhaps, described cell can transform with the cis acting element sequence by the mode that gene is knocked in, for example be incorporated into the strong composing type or the inducible promoter of the endogenous insulin gene upstream of described cell, with transformant with excessive generation and the secretion natural insulin.This can realize, because the upstream region of insulin gene is cloned (referring to Accession No.E00011, NM000207).In addition, described cell can use Recombulin gene (for example Accession No.J02547) to transform, and therefore makes described cell produce and the secretion Recombulin.

Therefore according to this aspect of the present invention, the pharmaceutical composition that is used to induce or quicken the agglutination of skin trauma or damage comprises, as active component, insulin secretory cell, and the pharmaceutically acceptable carrier that is designed for the local application of pharmaceutical composition.Advantageously, the insulin secretory cell that is administered to wound can form secretory granule, thereby secretes the insulin that is produced.Described insulin secretory cell can be endocrine cell.It can be the animal origin people source or the histocompatibility peopleization.Most preferably, described insulin secretory cell or conversion, or originate from body.By the excretory insulin of described insulin secretory cell insulin human or have the aminoacid sequence of insulin human preferably.Described insulin secretory cell can be a fibroblast, epithelial cell or keratinocyte, and prerequisite is to transform as mentioned above so that these cells produce and excreting insulin.

In another embodiment, the cell of skin trauma is transformed to produce and excreting insulin, to induce or to quicken the agglutination of skin trauma.

Therefore according to this aspect of the present invention, the pharmaceutical composition that is used to induce or quickens the agglutination of skin trauma comprises, as active component, be designed for and transform the nucleic acid construct of skin trauma cell with generation and excreting insulin, and the pharmaceutically acceptable carrier that is designed for the local application of pharmaceutical composition.

Above-mentioned any method for transformation, for example the construct with encoding insulin transforms, with containing the construct conversion that the mode that will knock in by gene is inserted into the cis acting element in endogenous insulin gene downstream, and transform generation and secretion to activate endogenous insulin with the construct of coding trans acting factor, may be used in embodiment of the present invention.

Because the vital stage is short, may break up and transformant that can't separating stable, be difficult to exogenous gene effectively be introduced primary cell with traditional method, thus before having hindered for the Research on effect of different PKC isotypes for skin.In order to overcome these obstacles, use viral vector to introduce interested gene.Viral genome is modified the viral vector that obtains the replication-defective virus form.The most widely used viral vector is retrovirus and adenovirus, and they can be used for experiment purpose and gene therapy purpose (13).Especially, compare with the reverse transcription carrier, the high efficiency of adenovirus infection in replication form cell not, the high titre of virus and the high expressed of transducin (transduced protein) make this system can be used for primary culture highly beneficially.Because the adenovirus unconformity is in host genome, stable virus titer can become replication defect type, so this virus formulation body causes dangerous minimum (14) of malignant tumor in the humans and animals model.Up to now, in skin, successfully used adenovirus construct in stripped and body, efficiently to infect (15,16) in the method.In this research, use a kind of adenovirus vector (17) of developing by I.Saito and his colleagues.It almost is adenovirus 5 genomes of total length that cosmid box (pAxCAwt) has, but does not contain E1A, and E1B and E3 zone make that this virus is replication defect type.It contains compound CAG promoter, and by the early stage immediately enhancer of cytomegalovirus, chicken beta-actin promoter and rabbit betaglobulin polyadenylation signal are formed, but the expression (13,17) of the DNA that induced strong is inserted.Interested gene is inserted in the cosmid box, then with adenovirus DNA terminal protein complex (TPC) cotransfection in the human embryonic kidney 293 cell.In 293 cells of expressing E1A and E1B zone, between cosmid box and adenovirus DNA-TPC, recombinate, with 100 times of required recombinant viruses of efficient generation of traditional method.This high efficiency mainly is owing to use adenovirus DNA-TPC to replace protein (proteinesed) DNA.And the existence in longer homology zone has increased the efficient of homologous recombination.Owing to have a plurality of EcoT221 site, avoided the regeneration of duplicating virus.Should be noted that keratinocyte is to infect with different PKC recombinant adenovirus, prove 24 hours after effective mistake expression PKC isotype (referring to embodiment 1).

Therefore, according to the present invention, the another kind of PKC of adjusting expresses and/or activatory method is by inducing PKC to cross expression in the skin trauma cell.This can be by realizing with cis acting element sequence transformant, thereby described cis acting element sequence makes described cell produce the native protein kinase c by the upstream that homologous recombination is incorporated into cell intrinsic protein kinase c.In addition, this also can be by realizing with recombiant protein kinase c gene transformation cell, such as but not limited to PKC-β 1 gene (Accession No.X06318, NM002738), PKC-β 2 genes (Accession No.X07109), PKC-γ gene (Accession No.L28035), PKC-θ gene (Accession No.L07032), PKC-λ gene (AccessionNo.D28577), PKC-τ gene (Accession No.L18964), PKC-α gene (Accession No.X52479), PKC-δ gene (Accession No.L07860, L07861), PKC-ε gene (Accession No.X72974), PKC-η gene (AccessionNo.Z15108) and PKC-ζ gene (Accession No.Z15108, X72973, NM002744), thus make described cell produce the recombiant protein kinase c.

According to this aspect of the present invention, the pharmaceutical composition that is used to induce or quickens the agglutination of skin trauma comprises, as active component, be designed for and transform the nucleic acid construct of skin trauma cell with the generation Protein kinase C, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

According to the present invention, the another kind of PKC of adjusting expresses and/or activatory method is to use the PKC activator, such as but not limited to Ca ²⁺, insulin or bryostatin 1 are realized, to induce or to quicken the agglutination of skin trauma.

Therefore according to this aspect of the invention, the pharmaceutical composition that is used to induce or quicken the agglutination of skin trauma or damage comprises, as active component, the PKC activator of treatment effective dose, inducing or to quicken the agglutination of skin trauma or damage, and pharmaceutically acceptable carrier.

According to the present invention, the another kind of PKC of adjusting expresses and/or activatory method is the expression and/or the activity of downward modulation PKC isotype.

The active downward modulation of PKC isotype can be used the pseudo-substrate isotype of PKC inhibitor, PKC α for example, the pseudo-substrate inhibitor (CalbioChem, California USA) of PKC ζ or PKC η, the for example medicinal peptide LY379196 of perhaps another kind of PKC isotype inhibitor (Elly Lilly, USA).

Perhaps, the active downward modulation of PKC isotype can realize with dominant (DN) PKC adenovirus construct, as described in following embodiment part.

The downward modulation of the expression of PKC isotype can be used siRNA (siRNA) molecule.It is one two step process that RNA disturbs.The first step, be defined as initial step, may be to pass through Dicer, the member's of the RNase III family of dsRNA specific ribonucleic acid enzyme effect, handle (cutting) dsRNA (directly introduce or by transgenic or virus) in the mode that depends on ATP, the dsRNA digestion of input is the siRNA (siRNA) of 21-23 nucleotide (nt).Follow-up cutting will be degraded to RNA the two strands (siRNA) of 19-21bp, and each all has 2 '-nucleotide, 3 ' jag [Hutvagner and Zamore Curr.Opin.Genetics andDevelopment 12:225-232 (2002); With Bernstein Nature 409:363-366 (2001)].

In the effect step, the double-stranded combination with the nuclease complex of siRNA forms the inductive silencing complex of RNA (RISC).The activation of RISC needs the siRNA two strands to untwist in the mode that ATP relies on.Active then RISC decides the homeodomain transcription thing by the interaction target of base pair, from 3 ' terminal fragment [the Hutvagner andZamore Curr.Opin.Genetics and Development 12:225-232 (2002) that mRNA is cut into 12 nucleotide of siRNA; Hammond et al. (2001) Nat.Rev.Gen.2:110-119 (2001); And SharpGenes.Dev.15:485-90 (2001)].Though the mechanism of cutting is still waiting to illustrate, and studies show that each RISC contains an one siRNA and RNAase[Hutvagner andZamore Curr.Opin.Genetics and Development 12:225-232 (2002)].

By the remarkable effectiveness of RNAi, show in the RNA i approach to have amplification step.Can be by the dsRNA of copy input, this can produce more siRNA, perhaps increases by the siRNA that duplicates formation.In addition or in addition, can realize amplification [Hammond et al.Nat.Rev.Gen.2:110-119 (2001), Sharp Genes.Dev.15:485-90 (2001) by many change event (multipleturnover events) of RISC; Hutvagnerand Zamore Curr.Opin.Genetics and Development 12:225-232 (2002)].Obtain more information, referring to following summary TuschlChemBiochem.2:239-245 (2001) about RNAi; Cullen Nat.Immunol.3:597-599 (2002); With Brantl Biochem.Biophys.Act.1575:15-25 (2002).

Can the synthetic RNAi molecule of the present invention that is applicable to as described below.At first, seek AA dinucleotide sequence in the downstream of the AUG start codon of PKC isotype mRNA sequence.The appearance meter record of each AA and near 3 ' 19 nucleotide is potential siRNA target site.Owing to more be rich in untranslated region (UTR) in the modulin binding site, preferred siRNA target site is selected from open reading frame.Conjugated protein and/or the translation initiation complex of UTR can disturb the combination [Tuschl ChemBiochem.2:239-245] of siRNA endonuclease multienzyme complex.Should be appreciated that the siRNA at untranslated region also is effectively, prove by GAPDH, wherein reduce at the siRNA mediated cell GAPDH mRNA of 5 ' UTR about 90%, destroyed fully protein level ( Www.ambion.com/techlib/tn/91/912.html).

Secondly, with any sequence alignment software, for example the BLAST software (www.ncbi.nlm.nih.gov/BLAST/) on the NCB I server is compared potential target site and suitable genome database (for example people, mice, rat etc.).Filter out the target site of inferring that has remarkable homology with other coded sequence.

Select qualified target sequence as the synthetic template of siRNA.Preferred sequence is to have those of low G/C content, and this is to be higher than 55% sequence specific energy mediated gene silencing more effectively mutually because this sequence has proved with those G/C content.Preferably select several target sites to estimate along the length of target gene.In order to estimate selected siRNA better, preferably use negative contrast simultaneously.Negative contrast siRNA preferably has same nucleotide composition with described siRNA, but does not have significant homology with genome.Therefore, preferably use the nucleotide sequence that mixes of siRNA, prerequisite is that it can not have any significant homology with any other gene.According to the present invention, suitable siRNA can be for example, can suppress the siRNA of PKC alpha expression, for example any nucleotide sequence of SEQ ID NO:1-6.

The another kind of reagent that can reduce the PKC isotype is to cut the mRNA transcript of PKC isotype or the DNA enzyme molecule of DNA sequence by specificity.The DNA enzyme is strand polynucleotide (Breaker, R.R.and Joyce, the G.Chemistry andBiology 1995 of energy cutting single-chain and double-stranded target sequence; 2:655; Santoro, S.W.﹠amp; Joyce, G.F.Proc.Natl.Acad.Sci.USA 1997; 943:4262).The universal model (" 10-23 " model) of DNA enzyme has been proposed." 10-23 " DNA enzyme has the catalyst structure domain of 15 deoxyribonucleotides, and flank is two substrate recognition structure territories that have seven to nine deoxyribonucleotides separately.Such DNA enzyme can be at purine: its substrate RNA (Santoro, S.W.﹠amp are effectively cut in the pyrimidine junction; Joyce, G.F.Proc.Natl.Acad.Sci.USA 199; The summary of DNA enzyme is referring to Khachigian, LM[Curr Opin Mol Ther4:119-21 (2002)]).

Make up and the example of synthetic, the engineered DNA enzyme that can discern strand and target cleavage site two strands that increases at the U.S.Pat.No.6 of Joyce etc., open in 326,174.That observes similar design recently suppresses the expression of urokinase receptor at the DNA enzyme of human urokinase receptor, and successfully suppress the interior colon cancer cell of body and shift (Itoh et al, 20002, Abstract 409, Ann Meeting Am Soc Gen Ther www.asgt.org).In another kind was used, the DNA enzyme that is complementary to bcr-ab1 oncogene successfully suppressed the expression of oncogene among the leukaemia, reduced the relapse rate of autologous bone marrow transplantation in CML and ALL.

The downward modulation of PKC isotype can also be used and can realize by special antisense polynucleotides of hybridizing with the mRNA transcript of the described PKC isotype of coding.

The design that can be used for effectively reducing the antisense molecule of PKC isotype will be considered two importances of antisense technology.First aspect is oligonucleotide sending in the Cytoplasm of suitable cell, and second aspect is the design that can specify the oligonucleotide of mRNA with the mode specific bond in cell that suppresses its translation.

Prior art has provided many delivery strategies, can be used for oligonucleotide effectively is delivered in the various kinds of cell type [referring to, Luft J Mol Med 76:75-6 (1998) for example; Kronenwett et al.Blood 91:852-62 (1998); Rajur et al.Bioconjug Chem 8:935-40 (1997); Lavigne et al.Biochem BiophysRes Commun 237:566-71 (1997) and Aoki et al. (1997) BiochemBiophys Res Commun 231:540-5 (1997)].

In addition, according to the thermodynamic cycle that can explain the energy of structural change in said target mrna and the oligonucleotide, differentiate the algorithm of sequence that has a binding affinity of the highest prediction with their said target mrna also be obtainable [referring to, for example, Walton et al.Biotechnol Bioeng65:1-9 (1999)].

These algorithms successfully are used for realizing the antisense method of cell.For example, the algorithm of Walton et al. exploitation makes scientist successfully design the antisense oligonucleotide of rabbit betaglobulin (RBG) and mouse tumor necrosis factor-alpha (TNF α) transcript.Same research group has been reported the antisense activity of oligonucleotide in cell culture at three model said target mrnas (people's lactic acid dehydrogenase A and B and rat gp130) choose reasonable recently, it, comprises with di-phosphate ester and the detection of thiophosphate oligonucleotide chemistry to three different targets in two kinds of cell types in effectively dynamically PCR method assessment almost all cases with proof.

In addition, several methods (Matveeva et al., Nature Biotechnology 16:1374-1375 (1998)) that design and predict the efficient of specific oligonucleotides with vitro system are also disclosed.

Several clinical experiments have proved that antisense oligonucleotide is safety, feasibility and activity.For example, the antisense oligonucleotide that is applicable to the treatment cancer has successfully used [Holmund et al., Curr Opin Mol Ther 1:372-85 (1999)], and decide the c-myb gene with target, the antisense strategy neoplastic hematologic disorder of p53 and Bcl-2 has also entered clinical experiment, has shown by patient's tolerance [Gerwitz Curr Opin Mol Ther 1:297-306 (1999)].

Recently, reported that the inhibition of human heparanase (heparanase) gene expression of antisense mediation in mouse model can suppress the pleura diffusion [Uno etal., Cancer Res 61:7855-60 (2001)] of human cancer cell.

Therefore, present common recognition is exactly the progress in the nearest antisense technology field, as mentioned above, cause having produced highly accurate antisense algorithm for design and multiple oligonucleotide delivery system, make those of ordinary skill to design and implement to be applicable to the downward modulation known array expression the antisense method and do not need over-drastic test and wrong experiment.

The another kind of reagent that can reduce the PKC isotype is ribozyme (ribozyme) molecule of the mRNA transcript of the special cutting encoded pkc isotype of energy.The sequence-specific that ribozyme is used for gene expression more and more suppresses, by the mRNA[Welchet al. of cutting coding proteins of interest, Curr Opin Biotechnol.9:486-96 (1998)].Can be designed for the ribozyme of any specific target RNA of cutting, this makes ribozyme become the of great value instrument in basic research and the treatment application.In the treatment field, ribozyme is used for hitting at infectious disease and decides viral RNA, in the hit oncogene of deciding advantage and of cancer in the genetic diseases special surely somatic variation [Welch et al., Clin Diagn Virol.10:163-71 (1998)] that hits.Ribozyme gene therapy scheme that it should be noted that most several H of being used for I V patients is tested carrying out the I phase.Recently, ribozyme is used to carry out Study on Transgenic Animal, the illustrating of the checking of gene target and approach.There are several ribozymes carrying out the clinical experiment of different phase.ANGIOZYME is the ribozyme in people's clinical and experimental study of being used in of first chemosynthesis.The ANGIOZYME specificity suppresses VEGF-r (vascular endothelial growth factor receptor), the formation of the key component in a kind of angiogenesis approach.Ribozyme Pharmaceuticals, Inc. and other company have proved the importance of angiogenesis inhibitor treatment in the animal model.HEPTAZYME, a kind of ribozyme that is designed for selective destruction hepatitis C virus (HCV) RNA has found effectively to reduce hepatitis C virus RNA (Ribozyme Pharmaceuticals, Incorporated-WEB homepage) in the detection of cell culture.

Preferably, the pharmaceutical composition that is used to induce or quicken the agglutination of skin trauma or damage further comprises at least a other reagent that is selected from the group that is made of following member: hormone, somatomedin, adipose cell hormone (adipokine), PKC δ RACK and GW9662.Suitable hormone includes, but are not limited to insulin.Suitable somatomedin can be but be not limited to interleukin-6 (IL-6), keratinocyte growth factor (KFG) or tumor necrosis factor (TNF α).Suitable adipose cell hormone can be but be not limited to adipsin or adiponectin (adiponectin).

When enforcement was of the present invention, the inventor found that surprisingly and unexpectedly copolymer-1 (glatiremar acetate) can significantly promote wound healing (referring to the embodiment 26 of following embodiment part) in vitro and in vivo.Known in the past copolymer-1 is a kind of immunomodulator, is used for the treatment of multiple sclerosis and central nervous system disease (U.S. Patent number 6,620,847,6,362,161,6,342,476,6,054,430,6,046,898,5,981,589 and 5,800,808; U. S. application serial number 10/615865,10/666857 and 10/014477), prior art is not described or is pointed out copolymer-1 to can be used for the accelerated wound healing process.

Therefore a kind of method that is used to induce or quicken the agglutination of skin trauma or damage is provided according to another aspect of the present invention, described method comprises that preferred concentration range for is 1 to 500 μ g/ml to the copolymer-1 of skin trauma or damage administering therapeutic effective dose.Therefore this aspect according to the present invention pharmaceutical composition of being used to implement this method comprises, as active component, the copolymer-1 and the pharmaceutically acceptable carrier of treatment effective dose.

Treatment/pharmacy activity component of the present invention can perhaps become pharmaceutical composition to be administered to wound with suitable carriers and/or mixed with excipients with itself.Be applicable to that pharmaceutical composition of the present invention comprises those compositionss of the active component that can obtain the expection therapeutic effect that wherein contains effective dose.

Here used " pharmaceutical composition " refers to one or more active component as herein described, protein, chemicals, nucleic acid or cell, or acceptable salt of its physiology or prodrug, with other chemical constituent conventional medicament for example, the preparation of physiology's suitable carriers and excipient.The purpose of pharmaceutical composition is to be convenient to give organism with chemical compound or cell.Pharmaceutical composition of the present invention can be with method preparation well known in the art, and for example by traditional mixing, dissolving is granulated, and sugar coating is pulverized, and capsule is made in emulsifying, embedding or freeze-drying process preparation.

Hereinafter, phrase " physiology's suitable carriers " and " pharmaceutically acceptable carrier " are used interchangeably, and all refer to not to cause significant stimulation and can not destroy the biological activity of conjugate (conjugate) of administration and the carrier or the diluent of character organism.

Term " excipient " is meant and adds in the pharmaceutical composition with the processing that further facilitates described active component and the inert substance of administration herein.The limiting examples of excipient comprises calcium carbonate, calcium phosphate, different sugar and dissimilar starch, cellulose derivative, gelatin, vegetable oil and Polyethylene Glycol.

The preparation of active component and medicine-feeding technology visible " Remington ' s PharmaceuticalSciences, " Mack Publishing Co., Easton, last version of PA is hereby incorporated by.

Although active component can as previously mentioned, be purpose of the present invention through different administrations, the topical mode is preferred, helps with topical carrier.Topical carrier is applicable to the topical of active component usually, comprises any this material well known in the art.For being made desired form, described compositions can select topical carrier, for example fluid or nonfluid carrier, and lotion, cream, paste, gel, powder, ointment, solvent, the fluid diluent, drop etc., and can comprise the material in naturally occurring or synthetic source.Obvious selected carrier can not produce harmful effect to active agent or other composition of topical formulations, should be stable for all the components in the topical formulations.The example that can be used for the suitable topical carrier of this paper comprises water, ethanol and other nontoxic organic solvent, glycerol, mineral oil, siloxanes, vaseline, lanoline, fatty acid, vegetable oil, p-Hydroxybenzoate, wax etc.Preferred formulation is colourless herein, tasteless ointment, fluid, washing liquid, frost and gel.

Ointment (ointment) is semi-solid preparation, is substrate with vaseline or other petroleum derivative typically.One skilled in the art will appreciate that specific ointment base to be used can provide best active component to send, other characteristic that needs and emollescence etc. for example preferably can be provided.The same with other carrier or vehicle, ointment base should be inert, stable, non-irritating not sensitization.As Remington:The Science and Practice ofPharmacy, point out in the 1399-1404 page or leaf of 19th Ed. (Easton, Pa.:Mack Publishing Co., 1995) that ointment base can be divided into four classes: oleaginous bases; Emulsifiable base; Emulsion matrix; And water-soluble base.The oil ointment base comprises, vegetable oil for example, and available from the fat of animal, and the semi-solid Hydrocarbon that obtains from oil.But the emulsifying ointment base is also referred to as and can absorbs ointment base, contains on a small quantity or not moisture, comprises hydroxy stearic acid sulfuric ester of glycerol for example, anhydrous lanolin and hydrophilic petrolatum (petrolatum).The emulsion ointment base is Water-In-Oil (W/O) or oil-in-water (O/W) emulsion, comprises, for example hexadecanol, glyceryl monostearate, lanoline and stearic acid.Preferred water-soluble ointment base prepares with different molecular weight polyethylene glycol; In addition, further information reference Remington:TheScience and Practice of Pharmacy.

Washing liquid is the preparation that is used for skin surface and need not rubs, normally fluid or semifluid preparation, and wherein solid particle comprises active agent, is present in water or the ethanol substrate.Washing liquid is solid suspension normally, can contain the fluid oil emulsion of oil-in-water type.Washing liquid is to be preferred for treating the large-area preparation of health herein, and this is to use easily owing to having more mobile compositions.Usually the insoluble matter in the washing liquid must grind.Washing liquid typically comprises suspension reagent disperseing better, and the chemical compound that is used to make described active agent localization and maintenance and contact skin, methylcellulose for example, sodium carboxymethyl cellulose etc.

The cream (creams) that contains selected active component as known in the art, is viscous fluid or semisolid emulsion, is oil-in-water or water in oil.Cream base can be washed, and contains oil phase, emulsifying agent and water.Oil phase is also referred to as " inside " phase sometimes, generally includes vaseline and aliphatic alcohol for example hexadecanol or octadecanol; Water is common, but optional, and volume surpasses oil phase, contains wetting agent usually.Emulsifying agent in the cream at Remington, above has explanation, and is normally non-ionic, anionic, cationic or amphoteric surfactant.

Gel preparation is preferably applied to scalp.Technical staff in the work of Topically active component preparation field knows that gel is semi-solid, the system of suspension type.Single-phase gels contains the organic macromolecule that is uniformly distributed in basically in the carrier fluid, aqueous typically, but preferably also contain ethanol and optional, oil.

Nucleic acid carrier includes but not limited to liposome, comprises the liposome that target is fixed, nucleic acid chelating agent, virus packets quilt etc.But also can use exposed nucleic acid to transform.

Can comprise in the topical formulations of the present invention and well known to a person skilled in the art various additives.For example, can use some active ingredient substance of dissolution with solvents.Other optional additive comprises skin penetration enhancer, opacifier, antioxidant, gellant, thickening agent and stabilizing agent etc.

Aforesaid, according to the present invention, the topical formulations that is used for the treatment of wound can contain other pharmaceutically active agent or the composition that tradition is used for the treatment of this wound.Comprise immunosuppressant, ciclosporin for example, antimetabolite, for example methotrexate, corticosteroid, vitamin D and novel vitamin D analogues, vitamin A or its analog, for example etretinate (etretinate), tar, coal tar, anti-itching and short keratinization (keratoplastic) reagent, for example cade oil, keratolysis reagent, salicylic acid for example, softening agent, lubricant, antiseptic and disinfectant, antibacterial leucoalizarin (being also referred to as anthralin) for example, photosensitizer, for example psoralen and methoxypsoralen and UV radiation.Can also add other reagent, for example antimicrobial reagent, antifungal agents, antibiotic and anti-inflammatory reagent.Can also carry out oxygen simultaneously and handle (high oxygen pressure).

Topical composition of the present invention can also be delivered on the skin with traditional transdermal patches or articles for use, and wherein said active ingredient compositions is contained in the layer structure, and wherein layer structure is fixed on the skin as drug delivery device.In a kind of like this structure, described active ingredient compositions is contained in one deck, or in " bank (reservoir) ", is surperficial bed course (upperbacking layer) above it.Layered structure can contain single bank, and perhaps it can contain a plurality of banks.In one embodiment, described bank contains the acceptable contact coherent substance of pharmacy of polymeric matrix, between the active component delivery period this system is fixed on the skin.The example of suitable skin contact coherent substance includes but not limited to polyethylene, polysiloxanes, polyisobutylene, polyacrylate and polyurethane etc.Specific active ingredient is depended in the selection of specific polymer adhesive, vehicle etc., and just, described adhesive must be compatible with all components that contains in the composition of active components.In addition, the bank that contains active component and contact skin adhesive should be independently with different layers, adhesive is under bank, in this case, bank can be aforesaid polymeric matrix, perhaps can be liquid or hydrogel reservoirs, perhaps can be some other form.

Bed course in these stratiform things is the surface layer as this device, as the primary structure element of layer structure, and gives this device with considerable elasticity.Selection should be all impermeable substantially with any other component that contains in the described composition of active components to described active component as the material of bed course, thereby prevents the surface layer forfeiture of any component by this device.Described bed course can be closure or inc, and this depends on whether wish skin aquation in the active component delivery process.Bed course preferably is made up of the thin slice or the film of preferred flexible elastomeric material.The example that is applicable to the polymer of bed course comprises polyethylene, polypropylene and polyester.

In storage process and before using, layered structure also comprises release liner.Remove this layer so that its basal surface from described device before using immediately, or active component bank or independently contact the adhesion layer exposure, make described system to be fixed on the skin.Described release liner should be made with the material of active component/carrier impermeable.

This device can be with conventional art manufacturing well known in the art, and for example with adhesive, the fluid mixture moulding of active component and carrier makes the release liner stratification then on bed course.Similarly, can with the adhesive mixture moulding on release liner, make the bed course stratification then.Perhaps, can under the situation that does not have active component or excipient, prepare the active component bank, be written in active component/carrier mixture by " soaking suction (soaking) " then.

The same with the topical formulations among the present invention, the active ingredient compositions that is contained in the active component bank of these layered systems can contain various ingredients.In some cases, described active component can be sent in the mode of " pure (neat) ", does not promptly contain other fluid.But under most of situation, the dissolving of described active component disperses or is suspended in suitable pharmaceutically acceptable carrier, typically in solvent or the gel.Other component that may contain comprises antiseptic, stabilizing agent and surfactant etc.

Pharmaceutical composition as herein described can also comprise suitable solid or gel phase carrier or excipient.The example of this carrier or excipient includes but not limited to calcium carbonate, calcium phosphate, and various sugar, starch, cellulose derivative, gelatin and polymer be Polyethylene Glycol for example.

Dosage depends on ailing type, the order of severity and the form of expression, and the patient is for the reaction of employed active component and dosage form, the effectiveness of specific conjugate and the surplus approach of employed administration.One skilled in the art can easily determine best dosage, dosed administration method and repetitive rate.Correct prescription, route of administration and dosage can be selected (referring to for example, Fingl, et al., 1975, in " The PharmacologicalBasis of Therapeutics ", Ch.1 p.1) according to patient's situation by each doctor.

Therefore, depend on the order of severity and the reactivity of the situation that will treat, dosage can be single or repeat administration, and treatment continues several days to several weeks, perhaps reduces until healing or skin trauma.

Use in the body or (cell) gene therapy technology that exsomatizes of the present invention in aspect some, it relates to the conversion that cell transformation and gene are knocked in type.Gene therapy used herein refers to interested hereditary material (for example DNA or RNA) is changed among the host to treat or to prevent genetic or acquired disease or situation or phenotype.The product that described interested hereditary material coding wishes to produce in vivo (for example protein, polypeptide, peptide, functional r NA, antisense RNA).For example, described interested hereditary material can be encoded and be had the hormone of therapeutic value, receptor, enzyme, polypeptide or peptide.Its summary generally can be referring to " Gene Therapy " (Advanced inPharmacology 40, Academic Press, 1997) original text.

Two kinds of basic skills of gene therapy are that exsomatize (1); (ii) vivo gene treatment.In the gene therapy of exsomatizing, from the patient, take out cell or derive cell, cultivate and at external treatment from the another kind source.Usually, with functional replacement gene by suitable gene delivery vector/method (transfection, transduction, homologous recombination etc.) and, in the time of if desired, expression system introducing cell makes the cell amplification of modification then and it is returned among host/patient in culture.Again the cell of implanting in these heredity has been presented at the hereditary material of expressed in situ transfection.

In the gene therapy, target cell is not taken out from the experimenter in vivo, but the hereditary material original position that will shift is just introduced in acceptor and accepted in the cell of organism.In another embodiment, if host gene is defective, described gene is repaired (Culver, 1998. (Abstract) Antisense DNA﹠amp in position; RNA basedtherapeutics, February 1998, Coronado, CA).The hereditary material that the cell that changes has been presented at the expressed in situ transfection is gone up in these heredity.

Described expression vector can be sent heterologous nucleic acids/transfer in the host cell.Described expression vector can comprise that to control the target of described nucleic acid in the mode of cell selective fixed, the element of expressing and transcribing, as known in the art.5 ' the UTR and/or the 3 ' UTR that it should be noted that common described gene can be replaced by 5 ' UTR of expression vector and/or 3 ' UTR.Therefore, expression vector as herein described is passable, if necessary, does not comprise the 5 ' UTR and/or the 3 ' UTR of the actual gene that will shift, and includes only specific aminoacid coding region.

Described expression vector can comprise the promoter of transcribing that is used to control described xenobiotic, described promoter can be composing type or induction type to allow alternative transcription.Randomly can comprise and obtain the necessary required enhancer of transcriptional level.The normally any DNA sequence of not translating of enhancer is closed on coded sequence and is worked (with cis) to change the basic transcription level that promoter is arranged.Described expression vector can also contain selection gene as mentioned below.

Carrier can be with any introducing cell or tissue in the several different methods well known in the art.These methods are at Sambrook et al., Molecular Cloning:A LaboratoryManual, Cold Springs Harbor Laboratory, New York 1989,1992), Ausubel et al., Current Protocols in Molecular Biology, JohnWiley and Sons, Baltimore, Maryland 1989), Chang et al.SomaticGene Therapy, CRC Press, Ann Arbor, MI 1995), Vega et al., GeneTargeting, CRC Press, Ann Arbor MI (995), Vectors:A Surveyof Molecular Cloning Vectors and Their Uses, Butterworths, Boston MA 1988) and among the Gilboa et al. (Biotechniques 4 (6): 504-512,1986) description is arranged, comprise, for example stable or temporary transfection, liposome transfection (lipofection), electroporation and use recombining virus carrier infection.In addition, referring to United States Patent (USP) 4,866, carrier that relates to the central nervous system in 042 and the positive and negative system of selection in the United States Patent (USP) 5,464,764 and 5,487,992.

Compare with other listed method and have several advantages by infect introducing nucleic acid.Because infection characterization thereby can obtain higher efficient.And virus is unusual specialization, typically can infect and breeds in specific cell type.Thereby their natural specificitys can be used for making carrier in vivo or at the mixed culture of tissue or the cell special surely cell type that hits.Viral vector can also be with special receptor or ligand modified to change the specificity of target by receptor-mediated incident.

The particular instance that is used to introduce with the dna viral vector of express recombinant sequence is the deutero-carrier A denop53TK of adenovirus.This vector expression herpesvirus thymine deoxyriboside kinase (TK) gene is selected and the expression cassette of desired recombination sequence so that forward or negative sense to be provided.This carrier can be used for infecting the cell with adenovirus receptor, comprises the tissue in most of epithelial origin and other source.This carrier and other carrier with similar desired function can be used for handling the cell of population mixture, comprise, for example cells in vitro or isolated culture are organized or people experimenter.

Can also comprise and be limited in the feature of expressing in the particular cell types.This feature comprises, for example, and promoter and the controlling element special to desirable cell type.

In addition, recombinant viral vector can be used for the expression in vivo of desirable nucleic acid, and this is because they for example can provide the laterally fixed specific advantage of infection and target.It is inherent in for example retroviral life cycle laterally infecting, and is that single infection cell produces many progeny virions, discharges and infect the process of adjacent cell.The result is that bulk zone is infected rapidly, wherein most of the infection by primary virion at first.This infection with vertical type is opposite, wherein infects reagent and only propagates by filial generation of future generation.Also can make can not horizontal transmission viral vector.If purpose is that specific gene is introduced a limited number of target cell, this feature may be useful.

As mentioned above, virus is the infection reagent of very specialization, can avoid host's defense mechanism in many cases.Typically, viral infection and in the cell of particular type, breeding.It is the natural specificity that has utilized it that the target of viral vector is decided specificity, and specific target is scheduled to cell type surely, thereby recombination is introduced in the infection cell.The carrier that uses in the method and composition of the present invention depends on the cell type that desired target is fixed, and those skilled in the art all know.

Can make up retroviral vector as infectious particles or just carry out initial one infection of taking turns.In first kind of situation, viral genome modified to make it keep all indispensable genes, regulating and controlling sequence and packaging signal are with synthetic new virus protein and RNA.These molecules are in case synthetic, and host cell can be packaged into new virion with described RNA, and it can carry out the infection of next round.Can also make the genome encoding of described carrier and express desirable recombination by genetic engineering.In not having infectious viral vector, the vector gene group is suddenlyd change usually and to destroy RNA is packaged into the necessary viral packaging signal of virion.Do not have such signal, the granule of any formation does not contain genome, therefore can not carry out next round and infect.The particular type of carrier depends on the application of expection.Actual carrier also is known, can obtain easily in the art, perhaps can be made up by the method for knowing by those skilled in the art.

Recombinant vector can several mode administrations.If the use viral vector, for example, the administration process can be utilized their target-specific, thereby not be used in the disease location topical.But topical can be treated more quickly and effectively.

Comprise in the body of the homologous recombination that the program of knocking in is used and described in isolated cells conversion process such as the following document, for example,

UnitedStates Patents 5,487,992,5, and 464,764,5,387,742,5,360,735,5,347,075,5,298,422,5,288,846,5,221,778,5,175,385,5,175,384,5,175,383,4,736,866 and Burkeand Olson, Methods in Enzymology, 194:251-2701991); Capecchi, Science 244:1288-1292 1989); Davies et al., Nucleic Acids Research, 20 (11) 2693-2698 1992); Dickinson et al., Human Molecular Genetics, 2 (8): 1299-13021993); Duff andLincoln, " Insertion of a pathogenic mutation into a yeast artificial chromosomecontaining the human APP gene and expression in ES cells ", Research Advances inAlzheimer ' s Disease and Related Disorders, 1995; Huxley et al., Genomics, 9:742-7501991); Jakobovits et al., Nature, 362:255-2611993); Lamb et al., Nature Genetics, 5:22-29 1993); Pearson and Choi, Proc.Natl.Acad.Sci.USA 1993) .90:10578-82; Rothstein, Methods in Enzymology, 194:281-3011991); Schedl et al., Nature, 362:258-261 1993); Strauss et al., Science, 259:1904-19071993).

Further, patent application WO 94/23049, W093/14200, WO 94/06908, and WO94/28123 also provides partial information.

Those of ordinary skills can know other purpose of the present invention apparently with reference to following non-restrictive example, advantage and new feature.In addition, above-mentioned all have experiment support in the following embodiments with each different embodiment of the present invention and the aspect described in the claim following claim part.

Embodiment

With reference to following embodiment, in nonrestrictive mode the present invention is illustrated with above-mentioned explanation.

Usually, experimental arrangement used among term used herein and the present invention comprises molecule, biochemistry, microorganism and recombinant DNA technology.These technology are existing in the literature to be explained in detail.Referring to, for example,

″Molecular?Cloning：A?Laboratory?Manual″Sambrook?etal.，(1989)；″Current?Protocols?in?Molecular?Biology″Volumes?I-III?Ausubel，R.M.，ed.(1994)；Ausubel?et?al.，″Current?Protocols?in?Molecular?Biology″，John?Wiley?andSons，Baltimore，Maryland(1989)；Perbal，″A?Practical?Guide?to?Molecular?Cloning″，John?Wiley&Sons，New?York(1988)；Watson?et?al.，″Recombinant?DNA″，ScientificAmerican?Books，New?York；Birren?et?al.(eds)″Genome?Analysis：A?LaboratoryManual?Series″，Vols.1-4，Cold?Spring?Harbor?Laboratory?Press，New?York(1998)；

Method as

U.S.Pat.Nos.4,666,828；4,683,202；4,801,531；5,192,659?and?5,272,057；″Cell?Biology：A?Laboratory?Handbook″，Volumes?I-IIICellis，J.E.，ed.(1994)；″Culture?of?Animal?Cells-A?Manual?of?Basic?Technique″byFreshney，Wiley-Liss，N.Y.(1994)，Third?Edition；″Current?Protocols?inImmunology″Volumes?I-III?Coligan?J.E.，ed.(1994)；Stites?et?al.(eds)，″Basic?andClinical?Immunology″(8th?Edition)，Appleton&Lange，Norwalk，CT(1994)；Mishelland?Shiigi(eds)，″Selected?Methods?in?Cellular?Immunology″，W.H.Freeman?andCo.，New?York(1980)；

Described in; Obtainable immunologic detection method extensively is described in patent and the scientific and technical literature, referring to, for example

U.S.Pat.Nos.3,791,932；3,839,153；3,850,752；3,850,578；3,853,987；3,867,517；3,879,262；3,901,654；3,935,074；3,984,533；3,996,345；4,034,074；4,098,876；4,879,219；5,011,771?and?5,281,521；″Olionucleotide?Synthesis″Gait，M.J.，ed.(1984)；“Nucleic?Acid?Hybridization″Hames，B.D.，and?Higgins?S.J.，eds.(1985)；″Transcription?and?Translation″Hames，B.D.，and?Higgins?S.J.，eds.(1984)；″Animal?Cell?Culture″Freshney，R.I.，ed.(1986)；″Immobilized?Cells?and?Enzymes″IRL?Press，(1986)；″A?Practical?Guideto?Molecular?Cloning″Perbal，B.，(1984)and″Methods?in?Enzymology″Vol.?1-317，Academic?Press；″PCR?Protocols：A?Guide?To?Methods?And?Applications″，AcademicPress，San?Diego，CA(1990)；Marshak?et?al.，″Strategies?for?Protein?Purification?andCharacterization-A?Laboratory?Course?Manual″CSHL?Press(1996)；

The full text that is incorporated herein all these documents as a reference.Other general reference also is provided herein.Wherein said program is well known in the art, provides for the reader is convenient.All information that wherein comprise all are incorporated herein by reference at this.

Material and experimental technique

Material: tissue culturing medium and serum purchase in Biological Industries (BeitHaEmek, Israel).Strengthen chemiluminescence (Enhanced Chemical Luminescence, ECL) manipulate the test kit of buying from BioRad (Israel).The antibody of monoclonal anti p-tyr purchase in Upstate Biotechnology Inc. (Lake Placid, NY, USA).The monoclonal of PKC isotype and polyclonal antibody purchase in Santa Cruz (California, USA) and Transduction Laboratories (Lexington, KY).α 6 rat anti-mouse mAb (GoH3) purchase in Pharmingen (San Diego, CA).The antibody 6844 of α 6A Cytoplasm domain derives from Dr.V.Quaranta (Scripps Research Institute, La Jolla, present CA).Rat mAb at the extracellular domain (346-11A) of mice β 4 derives from Dr.S.J.Kennel (Oak Ridge National Laboratory, OakRidge, present TN).At the rat mAB of phosphorylated tyrosine available from Sigma (St.Louis, MO), the anti-phosphorylation serine of rabbit available from Zymed (San Francisco, CA).Anti-rabbit of horseradish peroxidase and anti-mice IgG derive from Bio-Rad (Israel).Leupeptin, aprotinin, PMSF, DTT, Na-orthovanadate and Gastric inhibitory polypeptide available from Sigma Chemicals (St.Louis, MO).Insulin (humulin R-recombinant human insulin) available from EliLillyFrance SA (Fergersheim, France).IGF1 derives from the present of Cytolab (Israel).Keratin 14 antibody available from Babco-Convance (Richmond, CA).BDGF-BB is available from R﹠amp; D system (Minneapolis), the pseudo-substrate of the PKC α of myristoylation (myristolated) available from Calbinochem (San Diego, CA).

The separation of Mus keratinocyte and cultivation: described in document, separate former generation keratinocyte (18) from newborn skin.(Chelex-100 BioRad) cultivates keratinocyte among Eagle ' the s Minimal Essential Medium (EMEM) of the hyclone of Chu Liing at the Chelex that contains 8%.For the phenotype of the basal cell of keeping propagation, with final Ca ²⁺Concentration adjustment is 0.05mM.Experimentized by seven days in five days behind the bed board.

The preparation of cell extract and Western blot analyze: for obtaining the membrane component crude extract, containing 10 μ g/ml aprotiniies, 10 μ g/ml leupeptins, 2 μ g/ml Gastric inhibitory polypeptides (pepstatin), 1mM PMSF, 10mM EDTA, 200 μ M NaVO ₄With smudge cells among the PBS of 10mM NaF, prepare full cytolysis thing.After homogenate and freezing for 4 times/molten circulation, with solute place micro centrifuge with maximal rate 4 ℃ of rotations 20 minutes.The supernatant that will contain soluble cell solute protein component is transferred in another pipe.Precipitation is resuspended among the PBS that 250 μ l contain 1%Triton X-100 and protease and inhibitors of phosphatases,, in micro centrifuge, rotates with maximal rate at 4 ℃ 4 ℃ of insulations 30 minutes.Contain membrane component in the supernatant.Detect (Bio-Rad DC Protein AssayKit) with improved Lowery and measure protein concentration.The pair cell protein component carries out Westernblot analysis (6) described in document.

Be used for the preparation of the cytolysis thing of immunoprecipitation: the culture dish that contains keratinocyte is with not containing Ca ²⁺/ Mg ²⁺PBS washing.Make cell separation with mechanical force, place the mixture (20 μ g/ml leupeptins, the 10 μ g/ml aprotiniies that contain protease and inhibitors of phosphatases; 0.1mMPMSF; 1mM DTT; 200 μ M orthovanadates; 2 μ g/ml Gastric inhibitory polypeptides) RIPA buffer (50mM TrisHCl pH 7.4; 150mM NaCl; 1mM EDTA; 10mM NaF; 1%Tritonx100; 0.1%SDS, 1% NaTDC) in.With this prepared product 4 ℃ in micro centrifuge with maximum velocity centrifugation 20 minutes.Supernatant is used for immunoprecipitation.

Immunoprecipitation: (Santa Cruz, CA USA) mix, and suspension 4 ℃ of rotations 30 minutes continuously, is carried out the pretreatment of solute with 300 μ g cytolysis things and 25 μ l protein A/G Sepharose.Then with prepared product 4 ℃ with maximum velocity centrifugation 10 minutes, in supernatant, add 30 μ l A/G Sepharose and at indivedual antigenic specific polyclonals or monoclonal antibody (dilution rate 1: 100).Sample is spent the night 4 ℃ of rotations.Then with suspension 4 ℃ with maximum velocity centrifugation 10 minutes, will precipitate with the washing of RIPA buffer.Then with suspension with 15,000 * g recentrifuge (4 ℃, 10 minutes), with TBST washing four times.Add sample buffer (0.5M TrisHCl pH 6.8; 10%SDS; 10% glycerol; The 4%2-beta-mercaptoethanol; 0.05% bromophenol blue), sample was boiled 5 minutes, carry out SDS-PAGE then.

Adhere to detection: the stromatin with 20 μ g/ml among the PBS (Greiner) carries out coating (250 μ l/ hole) to 24 hole petri disses (petri plates), and 37 ℃ are incubated 1 hour.Be incubated the after scouring culture dish, and cultivate 30 minutes with the blocking-up non-specific binding in room temperature and 0.1%BSA.The keratinocyte culture is with the of short duration digestion of 0.25% trypsin, and is cell is resuspended after to be separated, with keratinocyte (1 * 10 ⁶) be added in the hole of coating, cultivated 1 hour at 37 ℃.Remove the cell that does not adhere to, the hole with PBS rinsing twice, is extracted remaining cell with 1M NaOH.Detect (Bio-Rad DCProtein Assay Kit) with improved Lowery and determine cell counting by protein concentration.The result is calculated as the percent with respect to untreated control.

Immunofluorescence: with former generation keratinocyte be inoculated on the glass slide of laminin (laminin) 5 coatings.With PKC adenovirus infection two the biggest keratinocytes one hour, use the PBS washed twice, be kept at low Ca ²⁺In the MEM culture of concentration.Infect the back twenty four hours; Cell is fixed 30 minutes with 4% paraformaldehyde, changed (permeabilization) thoroughly 5 minutes with 0.2%Triton then.For analyzing,, spend the night 4 ℃ of cultivations with the PKC antibody (Santa Cruz) that is diluted among the PBS that contains 1%BSA with the keratinocyte PBS rinsing of contrast and PKC infection.After the cultivation, microscope slide is used PBS washed twice 10 minutes, with two anti-the cultivations 20 minutes of biotinylated anti-rabbit, uses the PBS washed twice, cultivates 20 minutes with strepavidin-FITC.For analyzing the dyeing of alpha 6 beta 4, glass slide was handled on ice 5 minutes with 0.2%Triton X-100, in methanol, fix 5 minutes then.The antibody cultivation of microscope slide and anti-α 6 or anti-β 4 is spent the night, resist with two of biotinylated anti-rabbit respectively then and cultivated 20 minutes, use the PBS washed twice, cultivated 20 minutes with strepavidin-FITC.After the PBS washed twice, handle (mount) microscope slide with the glycerol buffer that contains 1% p-phenylenediamine (PPD) (Sigma), (MRC1024, Bio-Rad UK) detect fluorescence with the laser scanning co-focusing imaging microscope.

Adenovirus construct: recombinant adenoviral vector is as structure (19) as described in the document.The dominant negative mutant of mice PKC is to produce by the lysine residue that replaces ATP-binding site with alanine.With the cDNA of EcoR I from SRD expression vector cutting-out mutant, be connected on the pAxCA1w cosmid box, make up the Ax carrier.The active certificate of destruction of its autophosphorylation the dominant activity of described gene.

With PKC isotype gene transfer keratinocyte: the sucking-off culture medium, infected the keratinocyte culture one hour with the viral supernatant that contains the PKC recombinant adenovirus.Wash described culture twice with MEM then and cultivation again.Infected back ten hours, and cell transfer was not extremely contained the low Ca of serum ²⁺MEM in 24 hours.Keratinocyte from culture contrast and that handle with insulin or that handle with IGF1 is used to breed detection, ⁸⁶Rb absorbs, and perhaps extracts and is classified into cytosol and membrane component and carry out immunoprecipitation, immunofluorescence and Westernblotting.

PKC activity: in immunoprecipitate, measure special PKC activity with the keratinocyte culture prepared fresh after suitably handling.These cytolysis things are prepared in the RIPA buffer that does not contain NaF.(WI USA) measures activity, according to the description operation of manufacturer for Promega, Madison with SigmaTECT Protein Kinase C Assay System.In these researchs, use the pseudo-substrate of PKC α as substrate.

Cell proliferation: in 24 orifice plates, use [ ³H] thymidine mixes the mensuration cell proliferation.Cell usefulness [ ³H] thymidine (1 μ Ci/ml) pulse spends the night.Cultivate the back cell with PBS washing five times, 5%TCA was added each hole 30 minutes.Remove solution, cell is dissolved among the 1%Triton X-100.At the Tricarb liquid scintillation counter ³Thymidine to the labelling that mixes cell in the H window is counted.

Na ⁺/ K ⁺Pump activity: Na ⁺/ K ⁺The pump activity contains 2mM RbCl and 2.5 μ Ci by measuring full cell at 1ml ⁸⁶Rb's and do not contain K ⁺PBS in right ⁸⁶The absorption (uptake) of the ouabain sensitivity (ouabain-sensitive) of Rb is determined.Inhale after 15 minutes and go culture fluid to stop the absorption of Rb, then with cell cold 4 ℃ do not contain K ⁺PBS in fast rinsing four times, and be dissolved among the 1%Triton X-100.In scintillation vial, the cell on the culture dish is joined 3ml H ₂Among the O.At the Tricarb liquid scintillation counter ³In the H window sample is counted.Specifically with Na ⁺/ K ⁺It is to deduct by the absorption value that will measure under the condition that lacks inhibitor have 10 that the active relevant Rb of pump absorbs ^-4The cpm that accumulates under the condition of M ouabain determines.

PKC immunity kinase assay: purification with standardized PKC isozyme by Dr.P.Blumberg (NCI, NIH, U.S.) and Dr.Marcello G.Kazanietz (University of Pennsylvania, School of Medicine) generosity provide.Collect former generation keratinocyte in 500 μ l 1%Triton Lysis buffer (1%TritonX-100,10 μ g/ml aprotinin and leupeptins, 2 μ g/ml Gastric inhibitory polypeptides, 1mM PMSF, 1mM EDTA, 200 μ M Na ₂VO ₄, 10mM NaF is in 1x PBS) in.The molten product of born of the same parents was cultivated 30 minutes at 4 ℃, rotated 30 minutes with 16,000 * g at 4 ℃.Supernatant is gone in the fresh tube.Carry out immunoprecipitation at 4 ℃ of pair cell solutees and spend the night with the anti-α 6/GoH3 (PharMingen) of 5 a μ g/ sample and the protein A of 30 a μ l/ sample/G-plus agarose slurry (Santa Cruz).Pearl with the washing of RIPA buffer is once used 50mM Tris/HCl pH 7.5 washed twice.In detecting, each adds 35 μ l reaction buffer (1mM CaCl ₂, 20mMMgCl ₂, 50mM TrisHCl pH 7.5).Detect for each, in slurry, add 5.5 μ l/ the phospholipid capsule bubble suspensions that contain DMSO or 10mM TPA that detect, and the special PKC isozyme of normalized quantity.(1.25 μ Ci/ are detected [γ-32P] ATP, and Amersham) initial action continued 10 minutes at 30 ℃ to add 10 μ l/ the 125mM ATP that detect.Pearl RIPA buffer washed twice.(3xLaemmli 5%SDS), boiled sample 5 minutes in water-bath to add the protein application of sample dyestuff (proteinloading dye) of 30 a μ l/ sample.With SDS-PAGE isolated protein on 8.5% gel, transfer to Protran film (Schleicher﹠amp; Schuell) on, use the autoradiography video picture.The phosphorylation of the phosphorylation of histone and PKC peptide substrate is as the active contrast of PKC.

Experimental result

Embodiment 1

With the effective expression PKC isotype of crossing of recombinant adenoviral vector

Obtain high infection rate, the keratinocyte colony express recombinant protein of cultivation with reorganization beta galactosidase adenovirus above 90%.The adenovirus infection of reorganization beta galactosidase can not influence the viability or the cell growth of cell.Further, the expression of beta galactosidase lasts up to the cultivation in two weeks, can be used as contrast in the experiment below and infects.Detected the expression of reorganization PKC adenovirus construct induced protein and correct activatory efficient in mice keratinocyte culture.Shown in the Western blotting among Fig. 1, carried out with reorganization PKC adenovirus construct after 1 hour the infection 24 hours, observing specific PKC protein expression significantly increases, and is five to ten times of the endogenous expression of this specific isotype.Just can detect recombiant protein as far back as infecting in the keratinocyte culture that was infecting in back 6 hours, the peak value of expression obtained at 24 hours.Albumen continuous expression all in whole cultivation stage (reaching fortnight).

Embodiment 2

Crossing the PKC isotype of expressing is activated by the PKC activator

The recombiant protein of PKC isotype typically has reaction to the PKC activator.As shown in Figure 2, handle with bryostatin 1 and to induce PKC α and δ albumen to move to membrane component, and to PKC η and ζ isotype effect a little less than, also obtain analog result with endogenous isotype, as according to they to the needs of cofactor expect.

Embodiment 3

The native form of crossing the PKC isotype of expressing is activated

As far back as infecting back 18 hours, the PKC kinase assay just shows that the immunoprecipitate of different PKC isotypes does not need further to activate with the PKC activator just to have enzymatic activity (Fig. 3).

Embodiment 4

Cross the specific PKC isotype of expressing and in former generation keratinocyte, induce different metamorphosis

Each the PKC adenovirus construct that uses is all induced specific metamorphosis (Fig. 4) in former generation keratinocyte.Do not infect former generation mice keratinocyte culture and the cell that infects of beta galactosidase have the typical cubic morphology feature of the basal cell of the propagation in the culture.Regardless of the specificity of isotype, all PKC cross the expression keratinocyte and have shown the activatory typical metamorphosis of PKC, comprise cell elongation, the projection of neuron occurs.But each in the PKC isotype all has unique effect for the keratinocyte form.The infection induced keratinocyte stratification of PKC α has typical flat form.On the contrary, PKC η shows as the cell clone of deflation, is the morphological characteristic (Fig. 4) of the basal cell of fast breeding.As if two in the described isotype influence cellular matrix and cell and intercellular contact.Infected the back 18-48 hour at PKC δ, cell elongation, the projection of stretching out neuron.Be that cell is lost from culture dish gradually then, this takes place in incubation gradually.Cross the keratinocyte of expressing PKC ζ and show as circular keratinocyte bunch, its loosely is attached on the culture dish, little by little forfeiture in several days after infection.

Embodiment 5

Cross the difference location of the PKC isotype of expressing in the keratinocyte in former generation of infecting

Relevant by the metamorphosis that the immunofluorescence analysis proof is different with different celluar localizations.In the keratinocyte of propagation, PKC α, PKC δ and PKC ζ express in Cytoplasm and on the plasma membrane.Similar with the intrinsic protein expression, PKC η isotype is positioned the perinuclear region of cell territory (Fig. 5) of keratinocyte.PKC δ is relevant with the dynamic change of distribution with PKC ζ, and wherein after cell separation, the PKC isotype is expressed and mainly is positioned cell membrane (Fig. 5).

Embodiment 6

The regulation and control that the PKC isotype is expressed alpha 6 beta 4

Experimental result

Detected specific PKC isotype and regulated those proteic abilities for the substrate phenotypic characteristic of the basal layer of propagation.Because the downward modulation of alpha 6 beta 4 integrin is one of early stage incident that takes place in the keratinocyte atomization, assessed different PKC isotypes and regulated alpha 6 beta 4 integrins, a kind of integrin that is positioned the hemi desmosome of basal layer especially, the ability of expression.Immuning hybridization by Fig. 6 can be found out, compares with alpha 6 beta 4 integrin subunit level in the contrast keratinocyte, has only PKC δ and PKC ζ isotype can reduce alpha 6 beta 4 and expresses.Simultaneously, the level of α 3 or beta 1 integrin subunit does not reduce.On the contrary, as one man, the expression of crossing of PKC α isotype causes the alpha 6 beta 4 level to increase to two to three times (Fig. 6) that contrast is expressed.The expression of crossing of PKC η does not influence the proteic expression of alpha 6 beta 4.The cell that breaks up is relevant with several characteristic, and it comprises the reduction of growth rate after the downward modulation of alpha 6 beta 4 albumen, and new is keratic synthetic, cell separation, and forfeiture is adhered to basement membrane composition.The expression of crossing of not observing different PKC isotypes causes the keratin change of Expression.This comprises K5 and K14, characteristic body and the K1 and the K10 of substrate propagation keratinocyte, sour jujube shape differentiation (spinous differentiation) early stage characteristic body, expression.In addition, work as usefulness ³The H-thymidine mixes when analyzing growth rate, and expressing between forfeiture and the multiplication potentiality at alpha 6 beta 4 does not have dependency.

Embodiment 7

Crossing the PKC η and the PKC δ that express breeds at external evoked keratinocyte

The expression of crossing of PKC η and PKC δ significantly induces keratinocyte to breed (Fig. 7) to contrast five times of levels with twice respectively.PKC ζ and PKC α can not influence cell proliferation.

Embodiment 8

Crossing the PKC δ that expresses separates at external evoked keratinocyte with ζ

Studied the attachment characteristic that PKC δ and ζ cross the keratinocyte of expression.Compare with the contrast keratinocyte, comprise laminin 1 with specific stromatin, laminin 5, the adhesive ability of fibronectin and collagen protein do not change (data not shown).But, to express in the cell of PKC δ and PKC ζ isotype crossing, the forfeiture of the contact between cell and the culture dish and keratinocyte be separating from the culture dish relevant (Fig. 4) gradually.

Embodiment 9

The expression excessively of PKC isotype is to the hemi desmosome location influence of alpha 6 beta 4 integrin

Because the alpha 6 beta 4 expression is essential for the formation that hemi desmosome adheres to complex, has detected alpha 6 beta 4 downward modulation and cell separation and the relation of alpha 6 beta 4 between the location of hemi desmosome.Fig. 8 has shown the immunofluorescence analysis of the relation between alpha 6 beta 4 and the hemi desmosome complex.As shown in Figure 8, infect keratinocyte with contrast and compare, the rise that alpha 6 beta 4 integrin is expressed in crossing the keratinocyte (Fig. 6) of expressing PKC α is relevant to the increase of hemi desmosome complex integration with alpha 6 beta 4.Cross the cell of expressing PKC η and also induce combining of alpha 6 beta 4 integrin and hemi desmosome complex, though than spending observed lacking in the cell of expressing PKC α.As expected, cross the remarkable downward modulation of alpha 6 beta 4 integrin in the keratinocyte of expression and the minimizing relevant (Fig. 8) that alpha 6 beta 4 is integrated to cell hemi desmosome complex at PKC δ and PKC ζ.These results show that alpha 6 beta 4 integrin plays an important role in the grappling of following basement membrane in cell-matrix contact and keratinocyte.And the downward modulation of the alpha 6 beta 4 of PKC δ and ζ mediation makes keratinocyte begin to separate with a kind of approach that is different from the keratinocyte atomization.At last, for alpha 6 beta 4 downward modulation with the PKC mediation, the integration of the alpha 6 beta 4 on the hemi desmosome that reduces connects with separating of keratinocyte with specific metamorphosis, has detected and has adhered to the variation of amount of expressing the cell of different PKC isotypes with isolating mistake in the training period.In Fig. 9, the cell that adhered in to culture in 24 and 48 hours after the PKC adenovirus infection is counted.Can clear view lose at external evoked cell to PKC δ and PKC ζ.Simultaneously, the forfeiture of culture cell is relevant with the increase of buoyant cell in the culture medium.These results show that PKC δ is extremely important with cell differentiation and the early stage relevant separating step of migration for control with PKC ζ.

Embodiment 10

PKC η differentially regulates and control the propagation and the differentiation of keratinocyte under physiological condition.

From Fig. 7, can obviously find out, cross the speed propagation of cell express PKC η isotype,, also cultivate object height than crossing the keratinocyte of expressing other PKC isotype for five to seven times of the non-infected cells of contrast to quicken.But propagation induce the differentiation state that depends on keratinocyte, this is by regulating Ca in the culture medium ²⁺Concentration decision.Remaining on low Ca ²⁺Endogenous PKC η is positioned the perinuclear region of cell territory (Figure 10) of most of proliferative cells in the keratinocyte of the propagation of concentration (0.05mM).Under these conditions, the remarkable increase (Figure 11) of crossing induced expression keratinocyte propagation of PKC η.But, when with Ca ²⁺Concentration is increased to 0.12mM and makes keratinocyte differentiation time, and the crossing of PKC η expressed and do not induced propagation but further stimulate the keratinocyte differentiation.These results showed that expression PKC η only induced propagation in carrying out the physiological proliferating cells, and did not interfere the differentiation of cell.Also observe the difference of the regulating and controlling effect of PKC η expression in vivo.Differentiate the expression of PKC η in the skin of actively breeding and embryo's neuronal cell, and do not observe PKC η at sophisticated adult's brain, PKC η navigates to the granulosa of skin in epidermis.

Embodiment 11

Location and the cellular morphology of regulation and control PKC are especially expressed in crossing of PKC η and DNPKC η

For further conclusive evidence is supported the result that PKC η has positive role at the vegetative state or the differentiation state of keratinocyte, infected for the effect analysis of propagation and keratinocyte differentiation the effect of the dominant adenovirus PKC η construct of kinases inactivation by research.As shown in figure 12, the adenovirus infection at vegetative state and differentiation state PKC η and DNPKC η all is efficiently.As predicted DNPKC η induces the differentiation of keratinocyte in the keratinocyte of propagation, makes cellular morphology generation great variety comprise that cell becomes flat simultaneously like that, and the border between cell and the cell disappears, just with Ca ²⁺The form that inductive differentiation is followed changes similar (Figure 12 A-B).And, these variations be accompanied by keratinocyte propagation stop (Figure 11) and differentiation marker comprises keratin 1, keratin 10, significantly inducing of loricrin (loricrin) and fiber polymeric protein (filaggrin), its amount be increased to body in similar level (Figure 13 A-B) in the normal skin.Simultaneously, along with the beginning of differentiation program, the expression of crossing of DNPKC η does not destroy Ca ²⁺Inductive differentiation.These results show that PKC η and DNPKC η are used in propagation and the differentiation of differentially regulating and control keratinocyte under the physiological condition.

Embodiment 12

Experiment in the body

In order to detect the PKC η ability of regulating cell propagation and differentiation differentially in vivo, estimated the ability that PKC η induces wound (the full incisional wounds) healing that the nude mice back cuts fully.Verified the ability of keratinocyte expression external source recombiant protein with contrast β-gal adenovirus.As shown in figure 14, infect two weeks of back, skin continues that all β is arranged-the gal expression in external keratinocyte and body.What is interesting is when with contrast, when checking the wound healing process of mice after PKC α and the PKC η adenovirus construct local infection, to have only PKC η after local infection, just to induce granulation tissue to form in four days.This also comprises muscle, the organized formation of fat and skin corium.In the skin of contrast and PKC α infection, do not observe fine and close granulation tissue simultaneously, do not observe wound closure (Figure 14).Therefore, can be considered as be a main material standed for of regulation and control skin proliferation and differentiation in inducing wound healing process to PKC η.

Embodiment 13

Insulin special migration of inducing PKC δ in the keratinocyte of propagation

Found that two kinds of PKC isotypes of expressing in the skin influence the propagation of keratinocyte: PKC η and PKC δ.In order to attempt and differentiate the castle's intrinsic factor of the PKC isotype that activates specific adjusting skin proliferation, estimated the ability that mode that the somatomedin of several known promotion keratinocytes propagation relies on growth activates specific PKC isotype, having comprised: EGF, KGF, insulin, PDGF and IGF1.PKC isotype α, δ, ε, η and ζ express in skin.Because the activation of PKC isotype is relevant to the migration of membrane component with them, therefore checked the effect that these somatomedin move to film from cytosol for different PKC isotypes.As shown in figure 15, as far back as stimulating back 5 minutes, insulin moves to membrane component from Cytoplasm with regard to the special PKC of inducing δ.A few hours are kept in the expression of PKC δ on film behind the insulin stimulating.On the contrary, IGF1 has reduced the expression of PKC δ on film, and has increased its level relatively of expressing in cytoplasm fraction.The migration and the location that do not have other somatomedin appreciable impact PKC δ.Other PKC isotype is being comprised the later variation of all not observing in the distribution of IGF1 and insulin stimulating by any somatomedin.

Embodiment 14

Insulin special activation of inducing PKC δ in the keratinocyte of propagation

For whether the migration of determining PKC δ is enough to make its activation, measured kinase activity from the PKC immunoprecipitate of Cytoplasm and membrane component in the keratinocyte of insulin and IGF1 processing.As shown in figure 16, insulin rather than IGF1 increase the activity of PKC δ in the membrane component.In cytoplasm fraction, do not observe the raising of PKC alpha active.Insulin-induced activation is specific to PKC δ, is thirty minutes long all not observe PKC α ε, the activation of η or ζ behind insulin stimulating.Generally speaking, these results show the activation of insulin rather than I GF1 selective stimulating PKC δ.

Embodiment 15

Insulin and IGF1 have addition for keratinocyte propagation

Specific, activatedly whether mean insulin-induced mitogenesis approach specific in keratinocyte for what analyze PKC δ, induce by research insulin and IGF1 keratinocyte propagation efficiency test their mitogenesis effect, mix detection by thymidine.Shown in Figure 17 A, insulin and IGF1 stimulate thymidine to mix in dose-dependent mode, respectively 10 ^-7With 10 ^-8Reach the maximum value of inducing in the time of M.In each concentration, the maximal stimulus value of IGF1 is all than insulin height.What is interesting is, in all concentration, when two kinds of hormones use together, have add and mitogenesis effect (Figure 17 B).These results show that insulin is by propagation a kind of and the different approaches regulation and control keratinocyte that the inductive keratinocyte propagation of IGF1 is irrelevant.

Embodiment 16

Relation between insulin-induced PKC δ activation and the insulin-induced keratinocyte propagation

For the relation between the direct insulin-induced PKC δ of research activation and the insulin-induced keratinocyte propagation, cross the dominant negative mutant of the kinases inactivation of expression wild type PKC δ (WTPKC δ) and PKC with reorganization PKC adenovirus construct, it has destroyed endogenous PKC δ activity (DN PKC δ).Checked the effect of expression of crossing of WT PKC δ and DN PKC δ for insulin-induced keratinocyte propagation.Two constructs and PKC α construct can both efficiently express (Figure 18 A) in keratinocyte.And, infect can compare to exceed with PKC δ and PKC α and induce the specific PKC activity of isotype (Figure 18 B) several fold according to level.As expected, the PKC activity is not induced in the expression of crossing of DN PKC δ.Shown in Figure 19 A, the crossing of the WT PKC δ that non-transfected cells is handled with insulin or handled without insulin expressed and increased thymidine and be incorporated into approximately identical level, is untreated cell, or two to three times of the cell of transduceing with PKC α.And, in through the cell of expressing WT PKC δ, add insulin and can not cause any extra increase that thymidine mixes.IGF1 increases thymidine absorption (Figure 19 A) at the cell that does not infect similarly with crossing in the cell of expressing WT PKC δ and PKC α.Further participate in insulin-induced propagation directly by the activity proof PKC δ that destroys PKC δ.Shown in Figure 19 B, basic thymidine mixes slightly in crossing the cell express dominant PKC δ, but (significantly) significantly is lower than non-infected cells.Insulin-induced propagation has been eliminated in the expression of crossing of DN PKC δ fully, but does not influence the inductive propagation of IGF-1.And add and the effect of insulin and IGF1 reduces to the independent level of IGF1.

Embodiment 17

PKC δ activation is to the specificity of the approach of insulin-mediated

By research PKC δ and DN PKC δ for comprising IGF1 at multiple somatomedin, EGF, KGF, the mitogenic response of ECGF and PDGF be used for analyzing the specificity of PKC δ activation to the approach of insulin-mediated.As shown in figure 20, insulin-induced proliferation function is optionally eliminated in the expression of crossing of DN PKC δ, but does not block the effect of any other somatomedin that is detected.But the expression excessively of PKC δ is simulated insulin-induced propagation but is not influenced the inductive propagation of IGF1.The inductive propagation of the stimulation of EGF and KGF increases (Figure 21).These data show the propagation of insulin to the activation of the PKC δ approach mediation keratinocyte by relating to PKC δ, and this approach is positioned at EGF and KGF, the upstream of the signal transduction path of the two kinds of main known somatomedin that can regulate and control keratinocyte propagation.Generally speaking, find that insulin is the active specificity regulator of PKC δ, it may be at the regulation and control insulin, the specificity material standed in the inductive keratinocyte propagation of EGF and KGF.

Embodiment 18

The active keratinocyte propagation of insulin-induced PKC δ is by the transcriptional activation mediation of STAT 3

Further research PKC δ in the insulin signaling conduction effect and find inducing of transcriptional activation that it relates to the STAT3 mediation.As shown in figure 23, in former generation keratinocyte, PKC δ is special relevant with STAT3.Behind insulin stimulating, PKC δ is activated, then phosphorylation and activate STAT3 (Figure 24).And, the activation that the activity of destroying PKC δ with pharmacological inhibitor (kamaline) suppresses STAT3 with and appraise and decide the position.Further, as shown in figure 25, STAT3 crosses the similar propagation that induced expression and insulin-induced and PKC δ cross induced expression, crosses expression dominant PKC δ mutant and destroys PKC δ activity, has destroyed the ability that STAT3 induces keratinocyte to breed.These results show that totally insulin plays a role in the transcriptional activation relevant with keratinocyte propagation with PKC δ.

Embodiment 19

PKC δ and PKC ζ are essential for wound healing process in the body

With the invalid mice of isotype specificity PKC (PKC null mice) the checking PKC isotype importance in the wound healing process in vivo.Shown in Figure 22 A-B, when at PKC δ, PKC ζ, invalid mice (the gene knockout of PKC α, KO) and the back of the brood birth of their wild type Mus when causing the through thickness wound, in PKC δ and the invalid mice of PKC ζ, observe wound healing and postpone, and the invalid mice of PKC α does not have.This result shows or even is not having under the diabetes background that specific PKC isotype also is essential for the wound healing process of skin.

Embodiment 20

Be used for the single application of the insulin of wound healing in the body and repeatedly use

By cutting age in 8-10 week the C57BL mouse back cause wound, carry out following processing then: (i) in 7 days every day administration of insulin 0.1 μ M; (ii) in 7 days every day administration of insulin 1 μ M; (iii) in 7 days every day administration of insulin 10 μ M; (iv) causing back 4 days disposable employed insulin 1 μ M of wound; And (v) use carrier (PBS) contrast every day in 7 days.All mices are all causing wound execution in back seven days, measure the area of their open wound.As shown in figure 26, handle more effective than insulin processing every day significantly with insulin every day with the concentration of 1 μ M with lower (0.1 μ M) or higher (10 μ M) concentration.Be surprisingly, obviously more effective with the concentration single administration insulin of 1 μ M than use repetition processing in seven days every day with the insulin of same concentrations.

Because observed wound is covered by scar tissue, the formation of the actual closure of very difficult correct evaluation wound and the epidermis of reconstruction.Therefore determine the effect of insulin with Histological parameter for the closure of the epidermis of wound tissue and corium.The epidermis closure of wound be by with keratin 14 antibody (K14, Babco-Convance, Richmond, CA USA) determines that to wound section (sections) dyeing it can highlight the formation of basal cell on the wound breach.If the both sides of corium wound can both see in a visual field under the optical microscope that x100 doubly amplifies that then the corium closure of wound is considered as the positive.

As shown in figure 27, all insulins are handled and can both effectively be promoted epidermis and corium closure.Similar with result shown in Figure 26, handle significantly with the insulin of 1 μ M concentration every day and handle more effective every day than insulin with 0.1 μ M or 10 μ M concentration.In addition, obviously more effective with the insulin single administration of 1 μ M concentration than use repetition processing in seven days every day with the insulin of same concentrations.

Therefore, these results that obtained by morphology and Histological parameter obviously prove the therapeutic effect of insulin for wound healing in the body.These results show that surprisingly optimal number and/or the frequency of determining insulin administration are the committed steps of correctly treating wound.

Embodiment 21

Be used in combination insulin and platelet derived growth factor (PDGF-BB) and be used for wound healing in the body

Cause wound by cutting at 8-10 C57BL mouse back in age in week, cause wound and carry out following processing after 4 days: (i) carrier (PBS) contrast; (ii) insulin 1 μ M; (iii) PDGF-BB 10 μ M (R﹠amp; D Systems, Minneapolis, USA); (iv) insulin 1 μ M+PDGF-BB 10 μ M.Handle and put to death all mices in back three days, the wound of handling is analyzed epidermis and corium closure from the histology, described in above-mentioned embodiment 20.

As shown in figure 28, handling for epidermis closure (comparing according to high 30-40%) and corium closure (comparison is according to high 10-20%) with insulin or PDGF-BB separately all is that part is effective.But obviously higher epidermis closure (approximately comparison is according to high by 80%) and corium closure (about 60%) have been produced with insulin and PDGF-BB combined treatment.Therefore, these results show and are used in combination insulin and PDGF-BB can influence wound healing in synergistic mode.These results also show can be with insulin and other somatomedin or transforming factor EGF for example, and TGF β, KGF are used in combination the therapeutic treatment that is used for wound.

Embodiment 22

Be used in combination insulin and PKC alpha inhibitor and be used for wound healing in the body

Cause wound by cutting at 8-10 C57BL mouse back in age in week, with carrier (PBS) contrast or 0.67 μ M insulin (HO/01; Humulin, Eli Lilly is USA) with PKC alpha inhibitor (HO/02; The pseudo-substrate of the PKC α of myristoylation; Calibiochem, San Diego, CA, combination USA) was handled continuous 7 days every day.Cause wound and put to death all mices in back 7 days, processed wound is analyzed its wound closure, epidermis closure, the space differentiation of corium closure and epidermis cell.Measure open wound area to determine wound closure.If the both sides of corium wound can both see in a visual field under the optical microscope that x100 doubly amplifies that then the corium closure of wound is considered as the positive.With the k14 antibody of the formation that highlights basal cell on the wound breach to wound section dye to determine the epidermis closure of wound.The space differentiation of epidermis cell dyes to determine by with the k1 antibody of the epidermis cell that highlights new formation wound being cut into slices.

Shown in Figure 28-32, be used in combination insulin and (HO/01) significantly can promote wound closure (Figure 29 A-B), corium closure (Figure 30), the space differentiation (Figure 32) of epidermis closure (Figure 31) and epidermis cell with PKC alpha inhibitor (HO/02).As shown in figure 33, compare with vehicle Control, being used in combination insulin HO/01 and PKC alpha inhibitor HO/02 handles and can respectively wound epidermis closure be brought up to 70% from about 15%, the corium closure is brought up to 50% from about 15%, the space differentiation of epidermis cell is brought up to 50% from about 15%.

Therefore, these results show that being used in combination insulin and PKC alpha inhibitor carries out therapeutic treatment to wound and effectively promote the epidermis closure, corium closure, the space differentiation and the wound healing subsequently of epidermis cell.

Embodiment 23

Being used in combination insulin and PKC alpha inhibitor can prevent to use separately insulin to handle the bad side effect that causes

Cause wound by cutting at 8-10 C57BL mouse back in age in week, with carrier (PBS) contrast or 1 μ M insulin (Humulin, Eli Lilly, USA) or the pseudo-substrate (Calibiochem of 1 μ M insulin and 1 μ M PKC α, San Diego, CA, mixture USA) was handled continuous 7 days every day.Cause wound and put to death whole mices in back 7 days, to the multiplication capacity (PCNA) of processed wound from its epidermis of histology's analysis, angiogenesis, inflammation, the process of reconstruction of epidermis cell and wound breach.

Shown in following table 1, compare with the buffer contrast, only cause the significantly increase (being respectively 60% and 25%) that abnormal vascular generates in wound site with insulin processing meeting.Because wound healing process relates to the epidermis cell of fast breeding, so the increase of angiogenesis also may increase the danger of cause cancer development.On the other hand, when being used in combination insulin and PKC alpha inhibitor, do not observe angiogenesis in processed wound site.

Table 1

Use insulin separately and be used in combination the influence of insulin and PKC alpha inhibitor the order of severity of the angiogenesis of wound site

Handle	The multiplication capacity of epidermis (basal cell layer, PCNA)	Angiogenesis
			PBS contrasts (PBS processing) n=4	5786 average 6.5	Highly unusual normal

Handle	The multiplication capacity of epidermis (basal cell layer, PCNA)	Angiogenesis
			Only use insulin n=5	88655 average 6.4	Highly unusual normal highly unusually highly unusual
Insulin+PKC alpha inhibitor n=5	66423 average 4.2	Normal normal

In addition, only handle and can cause inflammation to increase with insulin, epidermal cell proliferation, the differentiation of epidermis cell spinous layer postpones and the cicatrix increase.When PKC alpha inhibitor and insulin are combined, do not observe and use insulin to handle the adverse side effect that causes separately.

Embodiment 24

The PKC alpha inhibitor reduces the wound inflammation

Site of injury is late period may to suppress agglutination with serious inflammatory reaction, prevents that therefore this inflammation from taking place to promote wound healing process.Correspondingly, checked the effect of PKC alpha inhibitor and insulin in the experiment below to the wound inflammation.

Cause wound by cutting at the C57BL mouse back, carried out following processing every day continuous 7 days: (i) PBS, contrast; (ii) 1 μ M PKC alpha inhibitor (the pseudo-substrate of myristoylation; Calibiochem, USA); (iii) 1 μ M insulin (Eli Lilly, USA); Or the mixture of 1 μ M PKC alpha inhibitor and 1 μ M insulin.Cause wound and put to death all mices in back 7 days, examine under a microscope the inflammation of processed wound.Incidence rate in the observed serious inflammation of wound site is summarised among the following table 1a.

Table 1a

Handle	The incidence rate (%) of serious inflammation in the wound
		The PBS contrast	??60.0
The PKC alpha inhibitor	??40.0

Handle	The incidence rate (%) of serious inflammation in the wound
		Insulin	??56.0
PKC alpha inhibitor+insulin	??50.0

The result shows compared with the control, uses the PKC alpha inhibitor to wound the incidence rate essence (33.3%) of severe trauma inflammation is reduced.Under experiment condition, use insulin not have the anti-inflammatory effect separately.

These results show that the PKC alpha inhibitor can be used for controlling the treatment of the serious inflammation of wound.The ability of the minimizing inflammation of certified PKC alpha inhibitor and it can promote the epidermis closure, and the ability (referring to the foregoing description 22) that corium space closed and epidermis cell breaks up makes it may become the most effective therapeutic agent of wound healing.

Embodiment 25

Regulate specific PKC isotype in the hypodermal cell expression and/or active with use the combined effect of different reagent to the outer wound closure of acceleration bodies to cell

Material and method:

Reagent: factor D (Adipsin) people, CalbioChem, California USA; Reorganization TNF α mice, R﹠amp; D Systems, Minneapolis USA; GW 9662, Caymanchemical, USA; The pseudo-substrate inhibitor of Protein kinase C α, CalbioChem, CaliforniaUSA; The pseudo-substrate inhibitor of Protein kinase C ζ, CalbioChem, California USA; The pseudo-substrate inhibitor of Protein kinase C η, CalbioChem, California USA; PDGF-BB, Cytolab, Israel; IL-6, Cytolab, Israel; KGF/FGF-7, Cytolab, Israel; IGF-1, Cytolab, Israel; TGF β 2, Cytolab, Israel; Epidermal growth factor (EGF), mice, Chemicon international, California USA; PKC δ RACK, Ana Spec, California USA; Rosiglitazone (Rosiglitazon), CalbioChem.California USA; Adiponectin (Adiponectin), MBL, Massachusetts USA and

TEVA.Israel.

External wound closure detects: in petri diss (5cm i.d.) keratinocyte and fibroblast (hypodermal cell) were cultivated five days, the head of pipette with 200 μ l forms artificial intersection cut in each culture dish then.Cultured cells is infected with the expression that can regulate specific PKC isotype and/or active adenovirus construct.Correspondingly, with the specific PKC of wild type (WT) PKC adenovirus construct activation, suppress specific PKC with dominant (DN) PKC adenovirus construct.In cultured cells, further add a kind of in the following reagent: insulin (6.7 * 10 ^-7M), adiponectin (every cultivation 1 μ g), adipsin (2 μ g/ml), IL-6 (every culture dish 1 μ g), GW9662 (every culture dish 1 μ g), KGF (every culture dish 1 μ g), TNF α (12 μ g/m l), TGF β, rosiglitazone, src inhibitor, PKC δ RACK (10 ^-7M) and the pseudo-substrate peptide for inhibiting (10 of PKC α ⁷M).After processing, determined the wound closure level that obtains in 24-48 hour, do not use from 0 (closed) and represent to the exponential quantity of 10 (closed fully).

The result:

Combined therapy is summarized in following table 2a-b and 3a-b to the effect of external fibroblast wound closure.The result shows that PKC alpha expression and/or active inhibition significantly promote wound closure (exponential quantity of wound closure is respectively 10 and 8) in the fibroblast when cooperating adipsin or the administration of insulin pair cell.The inhibition of the inhibition of PKC α and PKC η in fibroblast, the inhibition of PKC ε, the activation of PKC δ, or the combined also acceleration of wound of the activation of PKC ζ closure (exponential quantity of wound closure is respectively 9,9, and 9 and 7; Figure 34 A-E).In addition, in fibroblast the combined promotion wound healing of the administration of the inhibition of PKC ζ and KFG pair cell (the wound healing exponential quantity is 7; Figure 36).Further in addition, the inhibition of PKC β and insulin in the fibroblast, IL-6, the combined acceleration of wound closure of the administration of KGF or GW9662 (the wound closure exponential quantity is respectively 8,7, and 9 and 8; Figure 38 A-E).

Table 2a

Combined treatment is to the effect of external wound fibroblast closure ¹

	Contrast	??Adipsi??n	Insulin	??IL-6	??TGFβ	??KGF
							The inhibition of PKC α	??4	??10	??8	??3	??ND	??2
The inhibition of PKC β	??4	??ND	??8	??7	??1	??9
							The inhibition of PKC ζ	??2	??ND	??0	??2	??ND	??7

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

The ND=undetermined

Table 2b

Combined treatment is to the effect of external wound fibroblast closure ¹

	Adiponectin	??Rosiglita??zone	??GW9662	Src inhibitor
					The inhibition of PKC α	??3	??5	??2	??3
The inhibition of PKC β	??3	??3	??8	??2
					The inhibition of PKC ζ	??2	??2	??1	??1

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

Table 3a

Combined treatment is to the effect of external wound fibroblast closure ¹

	Contrast	The activation of PKCr η	The inhibition of PKC η	The activation of PKC ε
					The inhibition of PKC α	??4	5	9	?3

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

Table 3b

Combined treatment is to the effect of external wound fibroblast closure ¹

	The inhibition of PKC ε	The activation of PKC δ	The activation of PKC ζ	The inhibition of PKC ζ
					The inhibition of PKC α	?9	??9	??7	??1

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

Combined treatment is summarized among following table 4a-b and the 5a-b for the effect of external keratinocyte wound closure.The result shows when cooperating KGF, IL-6, in the time of TNF α or the administration of PKC δ RACK peptide pair cell, in the keratinocyte PKC alpha expression and/or active inhibition significantly promote wound closure (exponential quantity of wound closure is respectively 6,8,10 and 8; Figure 35 A-C and G).PKC η in the inhibition of PKC α and the cell in the keratinocyte, PKC ε, or the stimulation of PKC ζ is combined also increases wound closure (exponential quantity of wound closure is respectively 10,9 and 6; Figure 35 A, D-F and H).In addition, the inhibition of PKC ζ and IL-6 in the keratinocyte, (the wound closure exponential quantity is respectively 9,9 and 7 to the combined promotion wound closure of the administration of TNF-α or adiponectin pair cell; Figure 37 A-D).Further in addition, the activation of PKC ε in the activity of raising PKC δ and/or expression and the cell in keratinocyte, the activation of PKC ζ, or the inhibition of PKC α is combined, the perhaps cytotropic administration of adipsin, and (the wound closure exponential quantity is respectively 7 to the acceleration of wound closure, 8,8 and 8; Figure 39 A-E).

Table 4a

Combined treatment is to the effect of external wound keratinocyte closure ¹

	Contrast	??KGF	??IL-6	??TNFα	??Adipsi??n	Adiponectin
							Insulin	??ND	??ND	??ND	??ND	??ND	??ND
The inhibition of PKC α	??3	??6	??8	??10	??4	??2
							The activation of PKC δ	??ND	??ND	??ND	??ND	??8	??ND
The inhibition of PKC ζ	??0	??0	??9	??9	??ND	??7

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

The ND=undetermined

Table 4b

Combined treatment is to the effect of external wound keratinocyte closure ¹

	??Rosiglita??zone	Src inhibitor	??PKCδ??RACK	The pseudo-substrate of PKC α	Pseudo-substrate+the insulin of PKC α
						Insulin	??ND	??ND	??9	?9	??ND
The inhibition of PKC α	??2	??0	??8	?ND	??ND
						The activation of PKC β	??ND	??ND	??ND	?ND	??ND
The inhibition of PKC ζ	??2	??0	??0	?ND	??ND

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

The ND=undetermined

Table 5a

Combined treatment is to the effect of external wound keratinocyte closure ¹

	Contrast	The activation of PKC η	The inhibition of PKC η	The activation of PKC ε
					The inhibition of PKC α	??3	?10	?5	?9
The activation of PKC δ	??3	?ND	?ND	?7

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

The ND=undetermined

Table 5b

Combined treatment is to the effect of external wound keratinocyte closure ¹

	The inhibition of PKC ε	The activation of PKC ζ	The inhibition of PKC ζ	The inhibition of PKC α
					The inhibition of PKC α	?2	??6	??3
The activation of PKC δ	?3	??8	??3	??8

¹Closed numerical value is that 0 (not closed) is to 10 (closed fully)

The ND=undetermined

Therefore, this result shows as combined growth factor IL-6 for example, KGF, TNF α, hormone is insulin for example, the adipose cell hormone is adipsin or adiponectin for example, during the administration of PKC δ RACK and/or GW9662 pair cell, regulates the expression and/or the active significantly acceleration of wound closure of specific PKC isotype in the hypodermal cell of wound site and the epidermis cell.

Embodiment 26

Use copolymer-1 in the external and body and be used for wound healing

Material and method:

Copolymer-1: copolymer-1 (Glatiramer acetate) is a medicine

(Teva, active component Israel) are used for the treatment of multiple sclerosis clinically.Copolymer-1 is the synthetic polypeptide analog of myelin basic protein (MBP), and MBP is the natural component of myelin.Analyze from chemistry, copolymer-1 is L-glutamic acid and L-alanine, the acetate of the polymer of L-lysine and L-tyrosine.Its structural formula is: (Glu, Ala, Lys, Tyr) x.X CH3COOH (C5H9NO4C3H7NO2C6H14N2O2C9H11NO3) xxC2H4O 2.The mean molecule quantity of glatiramer acetate is 4,700-11,000 dalton.It synthetic is this four seed amino acid to be carried out chemical polymerization to form mean molecule quantity be 23,000 daltonian products (U.S. Patent No. 3,849,550).

Vitro detection: described in the foregoing description 24, detect substantially.Add copolymer-1 in the keratinocyte of cultivating, concentration is cultivated 55 μ g for each, adds separately or adds with pseudo-substrate (1 μ M) of PKC α and/or insulin (1 μ M).Handle and determined the wound closure level that obtains in back 48 hours, adopt exponential quantity from 0 (not closed) to 10 (closed fully).

Detect in the body: cause wound (20mm) by cutting at the C57BL mouse back, cause wound and carry out following processing after 4 days: (i) carrier (PBS) contrast; (ii) copolymer-1 (55 μ g/ml); The (iii) mixture of copolymer-1 (55 μ g/ml) and insulin (1 μ M); The (iv) mixture of pseudo-substrate peptide for inhibiting (1 μ M) of PKC α and insulin (1 μ M); And (v) copolymer-1 (55 μ g/ml), the mixture of pseudo-substrate peptide for inhibiting (1 μ M) of PKC α and insulin (1 μ M).From morphology, wound is assessed its (i) wound closure, the (ii) formation of crust and the (iii) hemorrhage/sepage of wound.

The result

Vitro detection: the keratinocyte of give cultivating is used copolymer-1 and is promoted external wound closure, and to be 0 (not closed) reach exponential quantity in the yardstick of 10 (closed fully) in the exponential quantity scope is 8.With the pseudo-substrate peptide for inhibiting combination of copolymer-1 and PKC α, or with the mixture combination of pseudo-substrate of PKC α and insulin, (the wound closure exponential quantity is respectively 8 and 9 to obtain similar effect; Figure 40 A-F).Therefore, these results show the significantly outer wound closure of acceleration bodies of copolymer-1 itself.

Detect in the body: compare with untreated contrast, use copolymer-1 separately for the wound of cutting, or with insulin and/or the pseudo-substrate combined administration of PKC α, can significantly reduce the formation of crust in wound breach zone and the acceleration of wound.In addition, the processing carried out of useful copolymer-1 all effectively prevent the hemorrhage and sepage of wound site.

Therefore, these results show to wound site use separately effective dose copolymer-1 or with the remarkable accelerated wound healing process of the pseudo-substrate peptide for inhibiting of insulin and/or PKC α combined administration.

Embodiment 27

The excretory material of thymus is for the influence of wound healing process

Material and method:

Back on normal grow up rodent or STZ diabetic mice (near the neck place) cuts out wound.Handle the back and put to death animal in 7 or 9 days,, analyze the epidermis and the corium closure of wound with the dyeing procedure described in the foregoing description 20 from the existence of histology near the thymus wound analysis wound site.

The result:

Shown in Figure 42 A-H, the epithelization of in the existence of wound breach close vicinity thymus and wound, quickening, the granulation of tissue is shunk relevant with corium.These observed results show that the excretory material of thymus can effectively be beneficial to the agglutination of wound.Correspondingly, thymus-derived material is thymosin for example, β thymosin (extrasin beta 4 for example, extrasin beta-10, extrasin beta 9, thymosin, extrasin beta 14), α thimosin (extrasin alpha for example, 1/zadaxin, prothymosin, parathymosin α), thymosin, IGFI, IGFII, NGF, Somat, Elityran, parathryoid hormone and/or thymosin peptide (THP) can be used for the treatment of accelerated wound healing process.

Be to be understood that the special characteristic of for clarity sake describing in the embodiment of separating of the present invention, also can make up in single embodiment provides.On the contrary, in single embodiment each feature of the present invention of describing for purpose of brevity, also can separately provide or provide with any suitable time combination.

Though the present invention describes in conjunction with specific embodiment, and multiple replacement scheme is clearly arranged to those skilled in the art, modifications and variations all are apparently.Correspondingly, the invention is intended to comprise all replacement schemes in the spirit and scope that drop on additional claim of the present invention, modifications and variations.All publications of mentioning in the description, patent, patent application is all introduced it in description as a reference in full, as pointing out each publication especially and separately, patent, patent application all is introduced into this paper as a reference.In addition, quoting or identifying and not should be understood to admit that these documents can be used as prior art of the present invention any list of references among the application.

List of references by Digital ID

(other document is quoted in the text)

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Sequence table

<110>Tennenbaum，Tamar

Sampson，Sanford

Kuroki，Toshio

Alt，Addy

Shen，Shlomzion

＜120〉be used for the method and the pharmaceutical composition of healing wounds

<130>28181

<160>16

<170>PatentIn?version?3.2

<210>1

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>1

ggaccacaaa?uucaucgcgt?t????21

<210>2

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉S iRNA oligonucleotide

<400>2

cgcgaugaau?uuguggucct?t????21

<210>3

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>3

auucaucgcg?cgcuucuuct?t????21

<210>4

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>4

gaagaagcgc?gcgaugaaut?t????21

<210>5

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>5

acaaggcuuc?cagugccaat?t????21

<210>6

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>6

uuggcacugg?aagccuugut?t????21

<210>7

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>7

ucccuaugga?uccaaacggt?t????21

<210>8

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>8

ccguuuggau?ccauagggat?t????21

<210>9

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>9

acuuauuccu?gaucccaagt?t????21

<210>10

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>10

cuugggauca?ggaauaagut?t????21

<210>11

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>11

auccgcagug?gaaugaguct?t????21

<210>12

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>12

gacucauucc?acugcggaut?t????21

<210>13

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>13

agaccgacga?cugucuguat?t????21

<210>14

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>14

uacagacagu?cgucggucut?t????21

<210>15

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>15

ggaaccacaa?gcaguauuct?t????21

<210>16

<211>21

<212>DNA/RNA

＜213〉artificial sequence

<220>

＜223〉SiRNA oligonucleotide

<400>16

gaauacugcu?ugugguucct?t????21

Claims (140)

1. the method for skin of inducing or quickening to damage or skin wound healing process, described method comprise to the insulin of the skin of described damage or skin trauma administering therapeutic effective dose and at least a other and the synergistic reagent of described insulin to induce or to quicken the skin of described damage or the described agglutination of skin trauma.

2. the process of claim 1 wherein that described using by single administration realize.

3. the process of claim 1 wherein that the insulin of described treatment effective dose has the insulin concentration of 0.1 μ M to 10 μ M.

4. the process of claim 1 wherein that described at least a other reagent is platelet derived growth factor.

5. the process of claim 1 wherein that described at least a other reagent is the PKC-alpha inhibitor.

6. the process of claim 1 wherein that described skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

7. the method for claim 6, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

8. the process of claim 1 wherein that described insulin recombinates.

9. the process of claim 1 wherein that described insulin is a natural origin.

10. the process of claim 1 wherein that described insulin and at least a other pack are contained in the pharmaceutical composition that is suitable for local application.

11. the method for claim 10, wherein said pharmaceutical composition is selected from aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersion liquid, ointment and ointment.

12. the method for claim 10, wherein said pharmaceutical composition comprises solid support.

13. the method for skin of inducing or quickening to damage or skin wound healing process, described method comprises the insulin secretory cell of implanted treatment effective dose in the skin of described damage or skin trauma, thereby induces or quicken the skin of described damage or the described agglutination of skin trauma.

14. the method for claim 13, wherein said skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

15. the method for claim 14, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

16. the method for claim 13, wherein said cell are transformed to produce and excreting insulin.

The PDX1 gene transformation 17. the method for claim 16, wherein said cell are recombinated, thus described cell is produced and the secretion natural insulin.

18. the method for claim 16, wherein said cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the endogenous insulin gene of described cell, thereby described cell produces and the secretion natural insulin.

19. the method for claim 16, wherein said cell are by the Recombulin gene transformation, thereby described cell produces and the secretion Recombulin.

20. the method for claim 13, wherein said insulin secretory cell can form secretory granule.

21. the method for claim 13, wherein said insulin secretory cell is endocrine cell.

22. the method for claim 13, wherein said insulin secretory cell are the people sources.

23. the method for claim 13, wherein said insulin secretory cell are histocompatibility peopleization animal origins.

24. the method for claim 13, wherein said insulin secretory cell secretion insulin human.

25. the method for claim 13, wherein said insulin secretory cell is an autogenous cell.

26. the method for claim 13, wherein said cell is selected from fibroblast, epithelial cell and keratinocyte.

27. method of inducing or quickening the agglutination of skin trauma, the cell that described method comprises the skin that transforms described damage or skin trauma to be producing and excreting insulin, thereby induces or quicken the skin of described damage or the described agglutination of skin trauma.

28. the method for claim 27, wherein said wound is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

29. the method for claim 28, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

The PDX1 gene transformation 30. the method for claim 27, wherein said cell are recombinated, thus described cell produces and the secretion natural insulin.

31. the method for claim 27, wherein said cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the endogenous insulin gene of described cell, thereby described cell produces and the secretion natural insulin.

32. the method for claim 27, wherein said cell are by the Recombulin gene transformation, thereby described cell produces and the secretion Recombulin.

33. the method for inducing or quickening the agglutination of skin trauma, described method comprise the cell that transforms described skin trauma with the generation Protein kinase C, thereby induce or quicken the skin of described damage or the described agglutination of skin trauma.

34. the method for claim 33, wherein said skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

35. the method for claim 34, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

36. the method for claim 33, wherein said cell are transformed with generation Protein kinase C transcriptional activator, thereby described cell produces the native protein kinase c.

37. the method for claim 33, wherein said cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the intrinsic protein kinase c of described cell, thereby described cell produces natural Protein kinase C.

38. the method for claim 33, wherein said cell are by the gene transformation of recombiant protein kinase c, thereby described cell produces the recombiant protein kinase c.

39. the method for claim 33, wherein said Protein kinase C is selected from PKC-β 1, PKC-β 2, PKC-γ, PKC-θ, PKC-λ and PKC-ι.

40. the method for claim 33, wherein said Protein kinase C is selected from PKC-α, PKC-δ, PKC-ε, PKC-η and PKC-ζ.

41. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, insulin as the treatment effective dose of active component, synergistic other reagent of at least a and described insulin, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

42. the pharmaceutical composition of claim 41, wherein said at least a other reagent is somatomedin.

43. the pharmaceutical composition of claim 42, wherein said somatomedin is a platelet derived growth factor.

44. the pharmaceutical composition of claim 41, wherein said at least a other reagent is the PKC-alpha inhibitor.

45. the pharmaceutical composition of claim 41, wherein said insulin is recombinated.

46. the pharmaceutical composition of claim 41, wherein said insulin is a natural origin.

47. the pharmaceutical composition of claim 41, wherein said wound is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

48. the method for claim 47, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

49. the pharmaceutical composition of claim 41, wherein said insulin and at least a other pack are contained in the preparation that is suitable for local application.

50. the pharmaceutical composition of claim 49, wherein said preparation is selected from aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersion liquid, ointment and ointment.

51. the pharmaceutical composition of claim 50, wherein said pharmaceutical composition comprises solid support.

52. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, as the insulin secretory cell of active component, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

53. the pharmaceutical composition of claim 52, wherein said wound is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

54. the method for claim 53, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

55. the pharmaceutical composition of claim 52, wherein said cell are transformed to produce and excreting insulin.

The PDX1 gene transformation 56. the pharmaceutical composition of claim 52, wherein said cell are recombinated, thus described cell produces and the secretion natural insulin.

57. the pharmaceutical composition of claim 52, wherein said cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the endogenous insulin gene of described cell, thereby described cell produces and the secretion natural insulin.

58. the pharmaceutical composition of claim 52, wherein said cell are by the Recombulin gene transformation, thereby described cell produces and the secretion Recombulin.

59. the pharmaceutical composition of claim 52, wherein said insulin secretory cell can form secretory granule.

60. the pharmaceutical composition of claim 52, wherein said insulin secretory cell is endocrine cell.

61. the pharmaceutical composition of claim 52, wherein said insulin secretory cell are the people sources.

62. the pharmaceutical composition of claim 52, wherein said insulin secretory cell are histocompatibility peopleization animal origins.

63. the pharmaceutical composition of claim 52, wherein said insulin secretory cell secretion insulin human.

64. the pharmaceutical composition of claim 52, wherein said insulin secretory cell is an autogenous cell.

65. the pharmaceutical composition of claim 52, wherein said cell is selected from fibroblast, epithelial cell and keratinocyte.

66. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, as active component, be designed for the nucleic acid construct of the cell of the described skin trauma of conversion with generation and excreting insulin, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

67. the pharmaceutical composition of claim 66, wherein said wound is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

68. the method for claim 67, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

The PDX1 gene transformation 69. the pharmaceutical composition of claim 66, wherein said cell are recombinated, thus described cell produces and the secretion natural insulin.

70. the pharmaceutical composition of claim 66, wherein said cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the endogenous insulin gene of described cell, thereby described cell produces and the secretion natural insulin.

71. the pharmaceutical composition of claim 66, wherein said cell are by the Recombulin gene transformation, thereby described cell produces and the secretion Recombulin.

72. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, as active component, be designed for the nucleic acid construct of the cell of the described skin trauma of conversion with the generation Protein kinase C, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

73. the pharmaceutical composition of claim 72, wherein said skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

74. the method for claim 73, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

75. the pharmaceutical composition of claim 72, wherein said cell are transformed with generation Protein kinase C transcriptional activator, thereby described cell produces the native protein kinase c.

76. the pharmaceutical composition of claim 72, wherein said cell is transformed by the cis acting element sequence, and this sequence is incorporated into the upstream of the intrinsic protein kinase c of described cell, thereby described cell produces natural Protein kinase C.

77. the pharmaceutical composition of claim 72, wherein said cell are by the gene transformation of recombiant protein kinase c, thereby described cell produces the recombiant protein kinase c.

78. the pharmaceutical composition of claim 72, wherein said Protein kinase C is selected from PKC-β 1, PKC-β 2, PKC-γ, PKC-θ, PKC-λ and PKC-ι.

79. the pharmaceutical composition of claim 72, wherein said Protein kinase C is selected from PKC-α, PKC-δ, PKC-ε, PKC-η and PKC-ζ.

80. the method for the agglutination of skin of inducing or quickening to damage or skin trauma, described method comprise that the adjusting PKC to described skin trauma administering therapeutic effective dose expresses or activatory reagent.

81. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprise, as active component, the adjusting PKC of treatment effective dose expresses or activatory reagent; And pharmaceutically acceptable carrier.

82. the method for the agglutination of skin of inducing or quickening to damage or skin trauma, described method comprises the PKC activator to the skin of described damage or skin trauma administering therapeutic effective dose, thereby induces or quicken the skin of described damage or the described agglutination of skin trauma.

83. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, as active component, the PKC activator of treatment effective dose, inducing or to quicken the skin of described damage or the described agglutination of skin trauma, and pharmaceutically acceptable carrier.

84. the method for inducing or quickening the isolated skin cell proliferation, described method comprise the reagent of the adjusting PKC generation that gives described Skin Cell effective dose.

85. the method for the agglutination of skin of inducing or quickening to damage or skin trauma, described method comprises to the skin of described damage or skin trauma uses the insulin of the treatment effective dose of single dose, thereby induces or quicken the skin of described damage or the described agglutination of skin trauma.

86. the method for claim 85, wherein said skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

87. the method for claim 86, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

88. the method for claim 85, wherein said insulin is recombinated.

89. the method for claim 85, wherein said insulin is a natural origin.

90. the method for claim 85, wherein said insulin is contained in the pharmaceutical composition that is suitable for local application.

91. the method for claim 90, wherein said pharmaceutical composition is selected from aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersion liquid, ointment and ointment.

92. the method for claim 90, wherein said pharmaceutical composition comprises solid support.

93. the method for inducing or quickening the agglutination of outmoded skin trauma, described method comprise the insulin of using the treatment effective dose of single dose to described outmoded skin trauma, thereby induce or quicken the described agglutination of described outmoded skin trauma.

94. the method for claim 93, wherein said outmoded skin trauma are at least 2 days ages.

95. the method for claim 93, wherein said outmoded skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

96. the method for claim 95, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

97. the method for claim 93, wherein said insulin is recombinated.

98. the method for claim 93, wherein said insulin is a natural origin.

99. the method for claim 93, wherein said insulin is contained in the pharmaceutical composition that is suitable for local application.

100. the method for claim 99, wherein said pharmaceutical composition is selected from aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersion liquid, ointment and ointment.

101. the method for claim 99, wherein said pharmaceutical composition comprises solid support.

102. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, as active component, the insulin of the single dose unit of the agglutination of the selected skin that can induce or quicken described damage or skin trauma, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

103. the pharmaceutical composition of claim 102, the insulin of wherein said single dose unit be in the described pharmaceutical composition of 0.01-0.2ml 0.001 to 5nM.

104. the pharmaceutical composition of claim 102, the insulin of wherein said single dose unit be in the described pharmaceutical composition of 0.01-0.2ml 0.01 to 0.5nM.

105. the pharmaceutical composition of claim 102, wherein said insulin is recombinated.

106. the pharmaceutical composition of claim 102, wherein said insulin is a natural origin.

107. the pharmaceutical composition of claim 102, the skin or the skin trauma of wherein said damage are selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

108. the method for claim 107, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

109. the pharmaceutical composition of claim 102, wherein said insulin is contained in the preparation that is suitable for local application.

110. the pharmaceutical composition of claim 109, wherein said preparation is selected from aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersion liquid, ointment and ointment.

111. the pharmaceutical composition of claim 102, wherein said pharmaceutical composition comprises solid support.

112. the method for the agglutination of skin of inducing or quickening to damage or skin trauma, described method comprises the copolymer-1 to described skin trauma administering therapeutic effective dose.

113. the method for claim 112, wherein said using by single administration undertaken.

114. the method for claim 111, the copolymer-1 of wherein said treatment effective dose are concentration is the copolymer-1 of 1 to 500 μ g/ml.

115. the method for claim 111, wherein said wound is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

116. the method for claim 115, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

117. the method for claim 111, wherein said basic protein analog is contained in the preparation that is suitable for local application.

118. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, as active component, the copolymer-1 of treatment effective dose, and the pharmaceutically acceptable carrier that is designed for described pharmaceutical composition local application.

119. the pharmaceutical composition of claim 118, wherein said skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

120. the method for claim 119, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

121. the pharmaceutical composition of claim 111, wherein said copolymer-1 is contained in the preparation that is suitable for local application.

122. the pharmaceutical composition of claim 121, wherein said preparation is selected from aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersion liquid, ointment and ointment.

123. the pharmaceutical composition of claim 111, wherein said pharmaceutical composition comprises solid support.

124. the method for the agglutination of skin of inducing or quickening to damage or skin trauma, described method comprise the expression and/or the activity of regulating at least a PKC isotype in the hypodermal cell that is settled in described skin trauma; Give at least a hormone that is selected from of described hypodermal cell administering therapeutic effective dose, somatomedin, the adipose cell hormone, other reagent of PKC δ RACK and GW9662 and the expression and/or the activity of regulating described PKC isotype, thus induce or quicken the skin of described damage or the described agglutination of skin trauma.

125. the method for claim 124, wherein said hypodermal cell are fibroblast or keratinocyte.

126. the method for claim 124, wherein said PKC isotype is selected from PKC-α, PKC-β, PKC-δ and PKC-ζ.

127. the method for claim 124, wherein said hormone is an insulin.

128. the method for claim 124, wherein said somatomedin is selected from IL-6, KFG and TNF α.

129. the method for claim 124, wherein said adipose cell hormone is adipsin or adiponectin.

130. the pharmaceutical composition of the agglutination of skin that is used to induce or quickens to damage or skin trauma, described pharmaceutical composition comprises, as active component, the expression or the activatory material of at least a PKC isotype of adjusting of treatment effective dose, with at least a hormone that is selected from, somatomedin, adipose cell hormone, other reagent of PKC δ RACK and GW9662, and pharmaceutically acceptable carrier.

131. the pharmaceutical composition of claim 130, wherein said hormone is an insulin.

132. the pharmaceutical composition of claim 130, wherein said somatomedin is selected from IL-6, KFG and TNF α.

133. the pharmaceutical composition of claim 130, wherein said adipose cell hormone is adipsin or adiponectin.

134. the pharmaceutical composition of claim 130, wherein said skin trauma is selected from ulcer, the wound that diabetes are relevant, burn, sunburn, aging skin wound, the cornea wound of festering, inflammatory gastroenteropathy wound, enteritis disease wound, the Crohn disease wound, ulcerative colitis, hemorrhoid, the epidermolysis surface disease wound of loosening, the skin wound of blistering, the psoriasis wound, animal skin wound, animal diabetic wound, the retinopathy wound, oral wounds (mucositis), encolpitis wound, gingival wound, laceration, operative incision wound and postoperative intestinal adhesion wound.

135. the pharmaceutical composition of claim 134, wherein said ulcer is selected from diabetic ulcer, decubital ulcer, venous ulcer, gastric ulcer and HIV related ulcers on.

136. the pharmaceutical composition of claim 130, it is contained in the preparation that is suitable for local application.

137. the pharmaceutical composition of claim 136, wherein said preparation is selected from aqueous solution, gel, cream, paste, lotion, spraying, suspension, powder, dispersion liquid, ointment and ointment.

138. the pharmaceutical composition of claim 130, wherein said pharmaceutical composition comprises solid support.

139. pharmaceutical composition that is suitable for local application with the agglutination of the skin of inducing or quickening to damage or skin trauma; described pharmaceutical composition comprises the insulin and synergistic other reagent of at least a and described insulin for the treatment of effective dose, and wherein said at least a other reagent is the pseudo-peptide substrate of myristoylation PKC α.

140. pharmaceutical composition that is suitable for local application with the agglutination of the skin of inducing or quickening to damage or skin trauma; described pharmaceutical composition is made up of insulin and synergistic other reagent of at least a and described insulin of treatment effective dose, and wherein said at least a other reagent is the pseudo-peptide substrate of myristoylation PKC α.

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