CN1453039A - Method of speeding wound repair and preventing complications - Google Patents
Method of speeding wound repair and preventing complications Download PDFInfo
- Publication number
- CN1453039A CN1453039A CN 02116900 CN02116900A CN1453039A CN 1453039 A CN1453039 A CN 1453039A CN 02116900 CN02116900 CN 02116900 CN 02116900 A CN02116900 A CN 02116900A CN 1453039 A CN1453039 A CN 1453039A
- Authority
- CN
- China
- Prior art keywords
- growth factor
- wound
- reparation
- cell growth
- liver cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention relates to biomedicine, and is especially the method of speeding repair of wound in tissue and organ and preventing complications. Human liver cell growth factor whole length coding region cDNA is inserted into self-constructed eukaryotic expression vector pUDK to obtain one recombinant plasmid, named pUDKH, carrying human liver cell growth factor, and the recombinant plasmid is compounded into injection or spray liquid. The injection or spray liquid is applied in one serial animal wound model tests, which proves its action of speeding heal of wound, preventing or reducing cicatrisation, promoting the regeneration of hair follicle and sebaceous gland, preventing adhesion, speeding the repair of nerves and skeletal muscles and promoting bone healing. Therefore, the treating method of applying recombinant plasmid mediated human liver cell growth factor has excellent application foreground in wound repair and complication treatment.
Description
The present invention relates to biomedical sector, the recombiant plasmid pUDKH that specifically relates to a kind of carrier's of application liver cell growth factor gene quickens the reparation of organ or tissue injury and prevents and treats the method for complication.
(hepatocyte growth factor HGF) is considered to a kind of hepatocyte mitogen to hepatocyte growth factor at first, finds afterwards that it to multiple histiocytic division, diffusion and form important regulatory role had taken place.Especially in the growth and regeneration of liver, HGF plays crucial effects, and HGF can treat hepatitis gravis, acute hepatitis, control hepatic fibrosis, liver cirrhosis and hepatocarcinoma.HGF also has very important therapeutical effect to kidney disease, can prevent and treat renal fibrosis, acute and chronic renal insufficiency etc.Studies show that HGF can promote the generation of pulmonary epithelial cells, the tube chamber formation of bronchi and the hypertrophy of pulmonary branches tracheal epithelial cell under physiological situation, lay a good foundation thereby treat illness such as pulmonary fibrosis, pneumonia for HGF.HGF promotes the effect of vascular endothelial proliferation to make it very likely be used for clinical treatment blood vessel wound and peripheral vascular disease, prevent that the coronary artery postoperative is narrow, prevent and treat arteriosclerosis with it, and can its anti-fibrosis effect and be used for the treatment of myocarditis and myocardial infarction.HGF also can promote neural generation, existence and regeneration, to the nutritious effect of nerve, can be used for prevention and treatment nerve injury.In addition, HGF all has certain preventive and therapeutic effect to peptic ulcer, bone articular cartilage disease etc.This shows that HGF is a very broad profile of cytokine of a biological activity, HGF will permeate every field (1) in clinical treatment with its multiple pharmacological effect.
Wound, burn comprise that the healing of operative incision remains a great problem of clinical existence, the healing of the difficult healing of wound such as healing, large-area burns or decubital ulcer of especially diabetes patient's wound healing, local radiotherapy wound, cause huge misery to the patient, also bring great burden, also do not have very effective method to promote the quickly-healing of these wounds at present to curative activity.HGF can promote effective, the high-quality healing of wound in theory from many aspects.At first, HGF can stimulate migration, the propagation of vascular endothelial cell, forms new vessels, thereby promotes the local blood circulation healing acceleration; But the motion of HGF chafe horn cell, propagation are impelled the cutization again of wound; HGF can significantly suppress the generation of TGF-β, and TGF-β be understand so far at most, with the closest cytokine of cicatrization, HGF is owing to can reduce the formation of cicatrix to the generation of anti-TGF-beta; HGF can strengthen the activity of collagenase in addition, impels the collagen of unnecessary formation to degrade effectively (3,4).
Studies show that in the past, the healing of fracture are extremely complicated processes, are under the effect of whole body and local factor, by the repair process of multiple tissue and factor participation.Between the cell and cell in osseous tissue, carry out mutual effect and adjusting between cell and the extracellular matrix, thereby finish the reparation and the reconstruction of bone.Fracture is the commonly encountered diseases and the frequently-occurring disease of surgical clinical, and the delayed union of fracture or bone does not connect connect body function, mental health and the normal social activity that has a strong impact on the patient.In recent years, revascularization in the knitting receives more and more closer concern, the popularity fracture has taken place in the blood vessel of fracture back bone stump, can not participate in the repair process of bone directly, it must be dredged, revascularization can promote osteanagenesis, and new vessels just has new osseous tissue to form after crossing over fracture region, and the mineralising of replacing bone crust also depends on revascularization in union of fracture.Therefore, seek a kind of cytokine that in fracture healing process, can promote revascularization, and it is local to be applied to fracture, will promotes the healing of fracturing, shorten the time of knitting.And HGF has proved the formation that can obviously promote new vessels, and HGF is the coupling factor of osteoblast and osteoclast, osteoblast and osteoclast all have the HGF receptor, HGF all can stimulate the propagation of the two, HGF can increase the osteoclast migration simultaneously, therefore osteoclast self is secreted activated HGF, thinks that HGF is to bone remodeling play an important role (5,6)
In the administration of HGF protein formulation, because of HGF in vivo the half-life shorter, thereby need heavy dose, repetitively administered just can keep local higher drug level; Yet, because of local or intravital zymolysis, even repeated application also often is difficult to reach valid density.Thereby the cost of application protein formulation is higher, and immature because of purifying process, brings side effect thereby easily produce ill effect to clinical treatment.A kind of new treatment pattern---the gene therapy methods of rising in recent years might overcome above-mentioned shortcoming.
The gene import system that gene therapy is used is broadly divided into non-virus carrier and viral vector two classes.The non-virus carrier method comprises naked DNA, liposome, particle gun etc.Especially directly be applied as method for optimizing with naked DNA in these methods.
In present clinical gene therapy, for reducing side reaction as far as possible, especially reduce the potential danger of gene mutation, gene is directly more and more favored with the form transfer of naked DNA.This method has following advantage: plasmid can carry long exogenous gene, the longest 48kb that reaches; DNA does not integrate with host cell chromosome, do not cause gene mutation, and expression time is of short duration relatively; Plasmid construction is simple, does not have infectivity and immunogenicity when the purified product quality reaches a standard again, is difficult for causing immunne response, but duplicate injection; Dividing and all can not express in the somatoblast.Shortcoming is that transfection efficiency is lower, but the HGF gene contains a secreting signal peptide, can be by the stronger biological effect of paracrine performance; And the low and of short duration expression of some specific disease naked DNA but is a big advantage, and safer when reaching therapeutic effect, be more suitable in clinical practice.
Viral vector can be retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, small virus or other virus.In these viruses, adenovirus preferentially is used for gene transfer.
In sum, how to quicken to organize or the reparation of organ wound and prevent and treat complication and be still a great problem in the medical science at present.Be not applied to clinical as yet and this one-to-many organ of HGF has the multifunctional cytokine of nutrition, repair function, for this reason, we have made up people HGF expression of gene plasmid pUDKH, after partial smearing or the injection, confirm that it has the effect of quickening the injuries of tissues and organs reparation and preventing and treating complication, thereby constitute a kind of new drug-supplying system and prevent and treat method, demonstrate potential clinical value.And the yield height of the pcDNA3-HGF fermentation back plasmid that more in the past made up of we constructed pUDKH, the high and HGF of transfection efficiency expresses height.
Show that from our experimental result pUDKH is the speed of accelerating wound obviously, the control tissue fibering suppresses the formation of pathologic scar.Our result has proved that using plasmid can effectively promote the quickly-healing of rabbit ear skin wound and promote hair follicle regeneration as carrier mediated HGF gene, promote sebaceous gland regeneration, promote the regeneration of hair and sebaceous gland, but have only in the mammal in people's the skin adnexa sweat gland is arranged, infer that therefore treatment that pUDKH is used for people's large-area burns may also can promote the regeneration of sweat gland.
We utilize HGF to promote biological functions such as new vessels forms, directed promotion wound healing, by the gene transfection technology, pUDKH is transferred to fracture local organization cell, make a large amount of secretion activity HGF of various kinds of cell such as osteoblast, myocyte, promote the hypertrophy of blood vessel and the reparation of wound, thereby quicken the healing of fracture.Clinically since the bone that wound, tumor post-operation and congenital bone malformation cause to carry out the patient of autologous bone transplanting after damaged quite a lot of, and union of fracture is identical with the mechanism of autologous bone transplanting reparation, so this project not only provides important scientific basis for HGF gene therapy fracture, and the reparation to promoting that clinically union of fracture and bone are damaged, also be extremely important.We are damaged with the facial bone of new zealand rabbit jaw to be example, proves that pUDKH can promote the healing of the facial bone injury of jaw, shortens the time of knitting, prevents the adhesion with surrounding tissue.
The result of our laboratory observation also shows, because of the dog femoral artery is made in the trouble limb that blood fortune seriously is interfered by ligation, not only skeletal muscle becomes because of tangible regression has taken place ischemia, amyotrophy occurs, unclear transverse striation of muscle fiber is clear, muscle fiber attenuates, ruptures, and adipose cell infiltration etc. is arranged in the muscle bundle; And significant pathological changes also taken place in femoral nerve, involves aixs cylinder, myelin and schwann's cell.And all obtained tangible recovery, the image that has even reached normal dogs through the muscle and the nerve of pUDKH processing dog limbs.
The result of our laboratory observation shows that also pUDKH has the promoting healing effect to the damage of new zealand rabbit mucous membrane of urinary bladder and smooth muscle, and the excessive formation of cicatrix afterwards that can prevent to heal.
In addition, the mediated by recombinant adenovirus HGF gene (Ad-HGF) that we use E1, E3 disappearance carries out the reparation research of wound, and the result shows that Ad-HGF has the effect of acceleration of wound reparation and complication prevention equally.
The object of the present invention is to provide a kind of method that is used for the acceleration of wound reparation and prevents and treats complication, this method is to utilize injection or the spray coating liquor of the recombiant plasmid pUDKH of carrier's liver cell growth factor gene to quicken the injuries of tissues and organs reparation and prevent and treat the generation of complication.
Specific embodiments of the present invention is as follows:
1. the liver cell growth factor gene of from the human placenta cDNA library, cloning people.
2. human hepatocyte growth factor coding region cDNA is inserted on the carrier pUDK that makes up voluntarily, obtains the plasmid pUDKH of carrier's liver cell growth factor gene expression cassette.
3. the preparation of plasmid pUDKH, purification and quality control.
4. observe this recombiant plasmid and can quicken union of wounded skin, and prevention of scar forms.
5. observe the growth that this recombiant plasmid can quicken skin follicle, hair, sebaceous gland.
6. observe recombiant plasmid nerve injury is had the acceleration repair.
7. observe recombiant plasmid muscle injury is had the acceleration repair.
8. observe recombiant plasmid bone injury is had the acceleration repair.
9. observe recombiant plasmid and can quicken mucous membrane of urinary bladder and level and smooth muscle injury reparation, and Film with Preventing Adhesion.
The invention provides a kind of method of quickening the injuries of tissues and organs reparation and preventing and treating complication, this method is to utilize the recombiant plasmid pUDKH of carrier's liver cell growth factor gene.This recombiant plasmid can be formulated into injection, baste etc. with liquid form to be used to quicken the injuries of tissues and organs reparation and to prevent and treat complication.
Use method provided by the invention, recombiant plasmid pUDKH control fiber disease with carrier's liver cell growth factor gene is to form mechanism from fiber to start with, in patient's body, import human hepatocyte growth factor gene, by generation that suppresses TGF β and the activity that strengthens extracellular matrix degrading enzyme such as collagenase, reduce level and the degrade collagen fiber of TGF β in the tissue, reach the purpose of control fiber disease.Therefore use method using recombinant plasmid control fiber disease ratio provided by the invention means of prevention in the past and have more science, curative effect is clearer and more definite, and is more thorough, and can reverse fibrotic processes.If the present invention is used, will provide a kind of effectively preventing means for the clinical treatment that does not still have at present the fiber disease of effective means of prevention.
Use method provided by the invention, recombiant plasmid pUDKH acceleration injuries of tissues and organs reparation with carrier's liver cell growth factor gene is from promoting angiogenesis in the damage part, promote hyperplasia, moving and start with, import human hepatocyte growth factor gene to damage location, by promoting vascularization in the part, promoting tissue regeneration to reach the purpose of accelerating tissue repair.Because of human hepatocyte growth factor is a multifunctional and nutritional reparative factor, can promote the reparation of histoorgan high-quality from many aspects, this therapy means of prevention more in the past has more science, and curative effect is clearer and more definite.
List of references
1. Liu Ke suffering. " hepatocyte proliferation factor (HGF) molecular medicine research latest developments ", the .2001 of Tianjin science tech publishing house, 1-140
2.Matsumoto?K，Hashimoto?K，Yushikawa?K，et?al.Markedstimulation?of?growthh?and?motility?of?human?keratinocytesby?hepatocyte?growth?factor.Exp?Cell?Res，1992；196：114-120
3.Ueki?T，Kaneda?Y，Tsutsui?H，et?al.Hepatocyte?growth?factorgene?therapy?of?liver?cirrhosis?in?rats.Nature?Med，1999，5：226
4.Zambonin?G，Camerino?C，Creco?G，et?al.Hydroxyapatitecoated?with?hpatocyte?growth?factor(HGF)stimulates?humanosteoblasts?in?vitro.Br?J?Bone?Joint?Surg?2000，82(13)：457
5.Grano?M，Galimi?F，Zambonin?G?et?al.Hepatocyte?gtowthfactor?is?a?coupling?factor?for?osteoclasts?and?osteoblastsin?vitro.Proc?Natl?Acad?Sci?USA，1996；93：7644
Embodiment: one, construction of recombinant plasmid, amplification and purification
1. construction of recombinant plasmid
(1) clone of human hepatocyte growth factor cDNA and sequencing
(Hepatocyte Growth Factor, HGF) cDNA sequential design primer, and introduce the BamHI restriction enzyme site in forward primer are introduced the SalI restriction enzyme site in the reverse primer to press the human hepatocyte growth factor of bibliographical information.The oligonucleotide sequence of forward primer is 5 '-ccatcgatgttaacatgtgggtgaccaaactc-3 ', the oligonucleotide sequence of reverse primer is 5 '-tgggatccgcggccgcctatgactgtggtacctt-3 ', from human placenta cDNA library's liver cell growth factor gene of cloning people.The PCR parameter is: 94 ℃ of 60sec, and 55 ℃ of 60sec, 72 ℃ of 90sec, period is 30,72 ℃ and extends 7min.The PCR product cloning to the BamHI and SalI restriction enzyme site of pBluescript SK (pSK) carrier, called after pSK-HGF.With XhoI enzyme action pSK-HGF, after agarose gel electrophoresis separates, reclaim the dna fragmentation of size about 3.5kb, 1.1kb, 0.6kb respectively, certainly get the 3.5kb fragment continuously pSK-HGF1 with the T4DNA ligase, again 1.1kb and 0.6kb fragment are inserted respectively in the XhoI restriction enzyme site of pSK carrier, respectively called after pSK-HGF2 and pSK-HGF3.All clone's process is seen Fig. 1-1.The sequencing reaction strategy is: pSK-HGF1 does a reaction with the T3 primer; PSK-HGF2 does a reaction respectively with T3 and T7 primer; PSK-HGF3 does a reaction with the T7 primer.Measure people HGF cDNA sequence with the two deoxidation cessation method of Sanger.
(2) structure of pUDK
The pUC18 cloning vehicle with Pvu II enzyme action, after agarose gel electrophoresis separates, is reclaimed the dna fragmentation of the about 2.3kb of size.With NruI and PvuII double digestion pcDNA3, reclaim the dna fragmentation of about 1kb, this fragment contains the CMV promoter, multiple clone site and growth hormone terminator.With these two fragments with the T4 dna ligase connect plasmid pUD (about 3.3kb).With AvaII and SspI double digestion pUD, reclaim the dna fragmentation of about 2.7kb, in addition with AvrII enzyme action pEGFP-N1, reclaim the dna fragmentation of about 1.1kb, this fragment contains complete kanamycin gene; Connect two recovery dna fragmentations and get plasmid pUDK (about 3.8kb).Make up sketch map and see Fig. 1.
(3) structure of the plasmid pUDKH of carrier's liver cell growth factor gene
With BamHI and ApaI double digestion pSK-HGF, reclaim the dna fragmentation of the about 2.2kb of size, be cloned into the multiple clone site (in two restriction enzyme sites of BamHI and ApaI) of pUDK carrier, obtain the recombinant plasmid vector of carrier's liver cell growth factor gene, called after pUDKH.Make up sketch map and see Fig. 1.
(4) preparation of the plasmid pUDKH of carrier's liver cell growth factor gene
Preparation flow comprises: fermentation, centrifugal collecting cell, alkaline denaturation cracking, separation and purification, quality testing.
(5) comparison of plasmid pUDKH and pcDNA3-HGF
With plasmid pUDKH and pcDNA3-HGF at the identical conditions bottom fermentation, the OD of the thalline 8 hours time of fermenting
600Value is respectively 1.6382 and 1.4308, through 42 ℃ of hot activations 1.5 hours, the OD of its thalline
600Value is respectively 1.6461 and 1.5167; The yield of plasmid pUDKH and pcDNA3-HGF is respectively 69.05 μ g/ml and 38.12 μ g/ml, and through 42 ℃ of hot activations 1.5 hours, its yield was respectively 78.83 μ g/ml and 59.33 μ g/ml.The rotaring redyeing 293 cell under identical conditions with plasmid pUDKH and pcDNA3-HGF, with the expression of HGF in the ELISA method detection supernatant, the result shows that the expression pUDKH in 293 cells is higher than pcDNA3-HGF.Two. recombiant plasmid Mediated Human liver cell growth factor gene (pUDKH) quickens the excessively effect of formation of union of wounded skin, prevention of scar
1. animal model
The female White Rabbit of New Zealand, body weight 2.5-3.5kg.The ear veutro does not have the trunk place shave hair sterilization after, with a trepan with microsurgery method excision holostrome skin until cartilage surface, the diameter 0.6cm of circular incision, every side 4-6 is individual.
2. gene transfer
Plasmid pUDKH with various dose in the time of excision skin is applied in the wound surface, and each wound respectively is coated with 10 μ g, 8 μ g, and 4 μ g, 2 μ g, 0 μ g only is coated with once.2 rabbits of each dosage group, totally 20 wounds.Dynamic observe wound healing speed after smearing, respectively the 8th day, the 12nd day with each wound of vernier caliper measurement do not heal transverse diameter, vertical footpath, calculate the area of the wound surface that do not heal.Smeared the back the 30th day, thick and to close on no site of injury skin holostrome thick with skin holostrome behind each wound healing of vernier caliper measurement, calculate loose index (Hypertrophic Index, HI) (HI=healing skin holostrome thick/close on the skin holostrome thick).To heal simultaneously wound surface and close on no site of injury skin holostrome (abdomen of ear, the back of the body both sides) and downcut in the lump through 10% formalin fixed, is done the routine paraffin wax section, row H.E. dyeing.Optical microscope down carries out the sxemiquantitative assessment according to the standard sketch map of keeping the score to the amount of each specimen fibrous tissue, be divided into 4 grades (+, ++, +++, ++ ++), observe the regeneration level of cutization again and cartilage simultaneously.
3. therapeutic evaluation
Experimental result shows, smearing the 8th day existing significant difference of healing rate in back, is coated with that 10 μ g and 8 μ g group all is coated with 4 μ g, 2 μ g group and the matched group healing is fast, and significant difference (p＜0.01) is arranged on the statistics; Though and 4 μ g, 2 μ g group and matched group comparison wound area dwindle but not statistically significant (p＞0.05).To smearing the back the 30th day, wound surface and contiguous no site of injury skin are downcut in the lump, do paraffin section after 10% formalin fixed, row H.E. dyeing.Observation by light microscope shows that it is light to be coated with pUDKH tissue slice keratinization of epidermis, the skin corium thin thickness, and the fibrous tissue amount is few; And the matched group fibrous tissue is many, the corium bed thickness.Fibrous tissue sxemiquantitative assessment result shows in the wound, and being coated with 10 μ g and 8 μ g group and being coated with between 4 μ g, 2 μ g group and matched group has significant difference (p＜0.01), and 4 μ g, 2 μ g organize and matched group does not have significant difference and be coated with.Calculate the loose index of each scar simultaneously, as an index of healing quality, loose index and cicatrix big or small proportional.The result shows that each group of smearing pUDKH is all little than the loose index of matched group, i.e. the formation of cicatrix is little, and outward appearance is more smooth, but only is coated with 10 μ g, and 8 μ g group relatively has significant difference (p＜0.01) with matched group.
In addition, be coated with 10 μ g, the growth of visible clump shape virgin wool on some healing skins of 8 μ g group, under the optical microscope visible hair follicle increase and the division of hair follicle cell mutually and sebaceous gland, and matched group and small dose group are not seen this phenomenon, show that the pUDKH that smears doses can promote the skin appendages growth.Three .pUDKH are to repair neural in the dog lower limb ischemia model, muscle
1. method
Hybrid dog (female, male both sexes, body weight 12-15kg) is in femoral artery in the left hind groin and is undertaken complete or half (making tube chamber be contracted to 7mm) ligation by the initiating terminal of external iliac artery bifurcation under intravenous injection pentobarbital anesthesia, causes the limb ischemia model.These model dogs are divided into matched group and pUDKH processed group at random.Promptly be engraved in the rectus femoris muscular tissue in the adductor longus of femoribus internus and the outside each minute 5 points behind the ligation femoral artery, each point is respectively injected 0.6 milliliter exposed blank plasmid pUDK or various dose (full ligation dog: 0.15,0.3, and 0.6mg/kg; Half hitch is pricked dog: the exposed plasmid pUDKH that 0.075,0.15, and 0.3mg/kg) carries the HGH gene.Postoperative in one's hands is taked muscle and femoral nerve tissue specimen from the hind leg of ligation in the time of full 3 months.These specimen are made section through conventional histopathology tabletting technology, observed result under the optical microscope.
2. result
(1) contrast dog
On longitudinal section, have only and still can indistinctly see its central authorities in some short sections of only a few nerve fiber and dyed caesious aixs cylinder, though all the other most nerve fibers then can't see aixs cylinder or have attenuate, engrain and be irregular crooked shape; The quantity of schwann's cell nuclear greatly reduces, and the nuclear that can see has all lost foamed structure, and shape also becomes the two ends point, examines slightly slightly or thin slightly some crooked fusiformis than normal, and it is deep mixed to dye; All having occurred many big and in irregular shape cavitys in the nearly all nerve fiber that more outstanding is arranges bunchiness mutually (myelin substrate after sustaining damage degraded has taken place, dissolved formed in conventional film-making process again), and the only even sections in indivedual nerve fibers of just often seen tiny cavity still can be seen, then all disappear at all the other positions, the dividing plate of the neural cutin (neurokerating) between the tiny cavity disappears mostly.The aixs cylinder of seeing most nerve fibers on the cross section has all lost the tone of shallow grey blue and has become engrain, thin and out-of-shape; Major part or the complete obiteration of the radial dividing plate of aixs cylinder periphery, and substituted by big and complete or imperfect belt clear area; The schwann's cell nuclear that is switched to also is the round state that contracts.The striped muscle of these side limbs has been observed myofibrosis, unclear transverse striation of muscle fiber is clear, and muscle fiber and nucleus attenuate, rupture, dye light, edema and appearance fat in the muscle bundle.
(2) pUDKH handles dog
The femoral nerve that dog is killed in work in 3 months also has bigger cavity on some sections of seeing a small part nerve fiber or some nerve fibers on the longitudinal section, but its quantity is far fewer than matched group, and most of sections of most of nerve fiber or some nerve fibers has all returned to the form and the regular neural cutin dividing plate of arranging of just often seen tiny cavity, can see the aixs cylinder of the blue color of light gray in the central.The quantity of schwann's cell nuclear is also more than this matched group, and only some is the fusiformis of engrain.Only seeing on the cross section that indivedual nerve fibers have annular clear area between aixs cylinder and schwann's cell film, and most nerve fiber has all returned to normal form, radial neural cutin dividing plate has been arranged in the periphery of aixs cylinder.The change that pUDKH handles dog hindlimb muscle tissue does not all have matched group obvious.Four .pUDKH are to the repair of rabbit mucous membrane of urinary bladder and level and smooth muscle injury
1. method
Prepare simple bladder muscle trauma model with 24-30 week new zealand rabbit, positive small incision exposes anterior wall of urinary bladder on the pubis, separates excision anterior wall of urinary bladder muscle layer tissue, 3 in preparation anterior wall of urinary bladder flesh break damage face, and area is 1cm
2Size.Apply simple solvent (A group) respectively, model control group (blank plasmid pUDK, B group) and experimental group (pUDKH, C group) solution only are coated with once.Postoperative was removed on the pubis and is managed in 4 days, went urethra to put pipe in 4-7 days; Carry out serial healing speed inspection, the cystography inspection is checked and histological examination substantially, and looser index (Hypertrophic index, HI).
2. result
20 rabbits of postoperative all survive to the experiment end.Postoperative is respectively organized the damaged wound surface of smooth muscle and is all reduced, and A, B and C form the face area, is respectively 44 ± 8mm in the time of 7 days
2, 43 ± 9mm
2With 28 ± 6mm
2(p＜0.05, Cvs A and B) is respectively 23 ± 7mm in the time of 14 days
2, 22 ± 6mm
2With 4 ± 4mm
2(p＜0.01, C vs A and B).Bladder image check and inspection discovery substantially respectively have a formation diverticulum with solvent treatment wound surface with Ad-GFP treatment wound surface, and all the other bladders all keep normal morphology.Postoperative healing bladder shows that A, B group shows that epithelial layer is normal, proliferation of fibrous tissue thickening under the mucosa and the flesh layer is thinner; C group has shown tangible three-decker, comprises that the normal vesical mucosa epithelium lining of form covers on the tela submucosa, is the level and smooth muscle bundle that one deck contains different shape thereupon, has identical normal histological structure with on every side tunica muscularis vesicae urinariae.Postoperative is in the time of 30 days, A, and the HI of B and C group is respectively 1.76 ± 0.17,1.83 ± 0.18 and 1.11 ± 0.18 (p＜0.05, C vsA and B).
3. conclusion
Experimental group rabbit bladder healing speed is fast, and gross anatomy inspection and cystography show that form is normal, and matched group wound surface smooth muscle of bladder reproduction speed is slow, and regeneration is incomplete, fibrous scar hypertrophy under the mucosa, and part mucosa evagination forms diverticulum.The recombiant plasmid of prompting carrier liver cell growth factor gene can promote the smooth muscle of bladder wound healing, reduces cicatrization.Five .pUDKH are to the repair of rabbit mandibular bone bone injury healing
1. method
20 of New Zealand's large ear rabbits (average weight 3kg) are divided into 5 groups at random, 4 every group.With 3% pentobarbital sodium (30mg/kg body weight) behind the auricular vein injecting anesthetic, dorsal position.Fixing limbs and head, head deflection right side.Left side jaw inferior segment preserved skin, sterilization.Make otch under the jaw of left side, be about 2.5cm, skin, subcutaneous tissue and platysma are cut in layering, and ligation arteria maxillaris externa, anterior facial vein are cut to surface of bone along the lower jaw lower edge, expose mandibular bone body posterior segment, do to peel off under cheek, the levum film with periosteal elevator.Making a diameter with the Buddha's warrior attendant bistrique at the lower jaw body then is that the connectivity bone of 0.5cm is damaged, will not fix.With 0.9% normal saline cleaning down wound surface, fully after the hemostasis, the bone defective region is clogged gelfoam, local injection pUDKH in gelfoam and in the cheek-tongue side soft tissue of defective region.The wound layering is tightly sewed up, the postoperative ad lib, and continuous three days intramuscular injection gentamycin 40,000 u, every day 1 time is with prevention infection.The 2nd, 4,6,8,12 weeks put to death animal in the operation back in batches, do fracture region angiography, stretching resistance, compressive resistance, anti-shearing force inspection, and histological examination, to judge the union of fracture situation.20 of matched group rabbits, the same substantially test group of grouping, processing and observation index, but topical application pUDK.
2. result
The result shows that pUDKH has the repair of quickening the healing of rabbit mandibular bone bone injury.Six. mediated by recombinant adenovirus human hepatocyte growth factor gene (Ad-HGF) is to the effect of repair in trauma
1. animal model
The female White Rabbit of New Zealand, body weight 2.5-3.5kg.The ear veutro does not have the blood vessel place shave hair sterilization after, until cartilage surface, the diameter of circular incision is 0.6cm to a trepan, every side 4-6 with microsurgery method excision holostrome skin.
2. effect assessment
In the time of excision skin recombinant adenovirus is applied in wound surface, smears 8ul (8.6 * 10
7Pfu/ μ l) (Ad-HGF group); The matched group wound is then smeared with dosage Ad-GFP.Postoperative downcuts wound surface and contiguous no site of injury skin January in the lump, does paraffin section, row HE dyeing.The result shows that smearing Ad-HGF group is coated with Ad-GFP to form the face healing fast, and inflammatory reaction is light, and the formation of cicatrix is little; HE dyeing shows that being coated with the Ad-HGF group is coated with that the Ad-GFP keratinization of epidermis is light, skin corium thickness is little, and fibrous tissue is thin.And can be observed the regeneration of hair follicle.
Claims (10)
1. a drug-supplying system and a Therapeutic Method that is used for the acceleration of wound reparation and prevents and treats complication is characterized in that this method is to utilize the recombiant plasmid injection of carrier's liver cell growth factor gene or spray coating liquor to be used for the acceleration of wound reparation and to prevent and treat complication.
2. an eukaryotic expression recombinant plasmid is characterized in that this recombiant plasmid carrier liver cell growth factor gene, contains the kalamycin resistance gene sequence; Liver cell growth factor gene is subjected to the polyadenylic acid signals-modulating of cytomegalovirus promoter, bovine growth hormone gene.The structure of this recombiant plasmid is:
PCMV: cytomegalovirus promoter; HGF: liver cell growth factor gene;
BGHpA: the polyadenylic acid of bovine growth hormone gene; Kana: kalamycin resistance gene;
ORI: origin of replication; PUDKH: carrier's liver cell growth factor gene recombiant plasmid
3. according to claim 1 and 2 described methods, it is characterized in that this method can quicken the reparation of skin trauma.
4. according to claim 1 and 2 described methods, it is characterized in that this method can quicken the Regeneration and Repair of skin appendages.
5. according to claim 1 and 2 described methods, it is characterized in that this method can prevent and treat cicatrix of skin and form.
6. according to claim 1 and 2 described methods, it is characterized in that this method can quicken the reparation and the control adhesion of injury of bladder.
7. according to claim 1 and 2 described methods, but it is characterized in that the reparation of this method accelerator nerve damage and prevent it and the adhesion of surrounding tissue.
8. according to claim 1 and 2 described methods, it is characterized in that this method can quicken the Regeneration and Repair of muscle injury.
9. according to claim 1 and 2 described methods, but it is characterized in that the reparation of this method accelerated bone damage.
10. according to claim 1 and 2 described methods, it is characterized in that this method can quicken the reparation of other organ or tissue's wound and prevent and treat the generation of complication.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021169004A CN100441226C (en) | 2002-04-26 | 2002-04-26 | Method of speeding wound repair and preventing complications |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021169004A CN100441226C (en) | 2002-04-26 | 2002-04-26 | Method of speeding wound repair and preventing complications |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1453039A true CN1453039A (en) | 2003-11-05 |
| CN100441226C CN100441226C (en) | 2008-12-10 |
Family
ID=29257039
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021169004A Expired - Lifetime CN100441226C (en) | 2002-04-26 | 2002-04-26 | Method of speeding wound repair and preventing complications |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100441226C (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100502942C (en) * | 2004-05-14 | 2009-06-24 | 中国人民解放军军事医学科学院放射医学研究所 | Application of liver cell growth factor in preventing and treating tissue adhesion |
| CN104013974A (en) * | 2014-06-11 | 2014-09-03 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain |
| CN113521309A (en) * | 2020-04-16 | 2021-10-22 | 中国人民解放军军事科学院军事医学研究院 | Application of human hepatocyte growth factor gene in eczema treatment and micro-needle medical instrument |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1284563A (en) * | 1999-08-17 | 2001-02-21 | 中国人民解放军军事医学科学院百环生物医学研究中心 | Recombined carrier carrying human liver cell growth factor gene and its application in treating ischemic diseases |
| CN1142272C (en) * | 2000-02-02 | 2004-03-17 | 中国人民解放军军事医学科学院百环生物医学研究中心 | Rcombinant adenovirus and its use in preventing and treating fibrous diseases |
| CN1150035C (en) * | 2000-12-21 | 2004-05-19 | 中国人民解放军军事医学科学院放射医学研究所 | Recombination plasmid and application in disease prevention and control |
-
2002
- 2002-04-26 CN CNB021169004A patent/CN100441226C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100502942C (en) * | 2004-05-14 | 2009-06-24 | 中国人民解放军军事医学科学院放射医学研究所 | Application of liver cell growth factor in preventing and treating tissue adhesion |
| CN104013974A (en) * | 2014-06-11 | 2014-09-03 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain |
| CN113521309A (en) * | 2020-04-16 | 2021-10-22 | 中国人民解放军军事科学院军事医学研究院 | Application of human hepatocyte growth factor gene in eczema treatment and micro-needle medical instrument |
| CN113521309B (en) * | 2020-04-16 | 2023-07-07 | 中国人民解放军军事科学院军事医学研究院 | Application of human hepatocyte growth factor gene in treatment of eczema and microneedle medicine instrument |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100441226C (en) | 2008-12-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Current methods for skeletal muscle tissue repair and regeneration | |
| RU2542391C2 (en) | Method for neogenesis | |
| EP2327428A2 (en) | Pharmaceutical compositions for repairing or replacing damaged tissues, and for improving myocardial infarction | |
| Sun et al. | Conduits harnessing spatially controlled cell-secreted neurotrophic factors improve peripheral nerve regeneration | |
| CN104854129B (en) | Use of pigment epithelium derived factor derived polypeptide for promoting muscle or tendon regeneration or arterial angiogenesis | |
| CN1668734A (en) | Method and device for multiplying and differentiating cells in the presence of growth factors and of a biological matrix or of a supporting structure | |
| TW201315479A (en) | Pharmaceutical composition for promoting arteriogenesis, and preparation method and applications for the same | |
| Qiao et al. | Synergistic effects of adipose-derived stem cells combined with decellularized myocardial matrix on the treatment of myocardial infarction in rats | |
| US20190070337A1 (en) | Multilayered cell sheet of neural crest stem cells and method of preparing the same | |
| CN104056258A (en) | Composition for promoting physiologically regulative regeneration of damaged tissue as well as preparation method and use thereof | |
| US20080102059A1 (en) | Treatment for arthritis | |
| Tian et al. | Repair of urethral defects by an adipose mesenchymal stem cell‑porous silk fibroin material | |
| CN105263507A (en) | The use of sdf-1 to mitigate scar formation | |
| Zhang et al. | Velvet antler peptides reduce scarring via inhibiting the TGF-β signaling pathway during wound healing | |
| CN111450119A (en) | Perinatal tissue-derived extracellular matrix hydrogel preparation for promoting organ injury repair | |
| CN100441226C (en) | Method of speeding wound repair and preventing complications | |
| CN112843229A (en) | Application of medicine for promoting skin injury repair in plastic surgery repair | |
| CN1150035C (en) | Recombination plasmid and application in disease prevention and control | |
| CN111405898B (en) | Method for destroying cell mechanical homeostasis and promoting tissue and organ regeneration and repair and application thereof | |
| CN114869841B (en) | Microneedle patch carrying stem cell active biological factor and large-dose triamcinolone acetonide and preparation method thereof | |
| CN104013974A (en) | Application of hepatocyte growth factor gene in preparation of medicament for preventing and/or treating pain | |
| Li et al. | Nerve growth factor-basic fibroblast growth factor poly-lactide co-glycolid sustained-release microspheres and the small gap sleeve bridging technique to repair peripheral nerve injury | |
| Firmansyah et al. | Unraveling the significance of growth factors (TGF-β, PDGF, KGF, FGF, Pro Collagen, VEGF) in the Dynamic of Wound Healing | |
| KR101561744B1 (en) | 3D cell implant patch and process for preparing the same | |
| CN1250287C (en) | Application of recombinant adenovirus in treating cerebral ischemia, traumatic restoration and tissue adhesion |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| ASS | Succession or assignment of patent right |
Owner name: WUHAN OPTICAL VALLEY RENFU BIOPHARMACEUTICAL CO., Free format text: FORMER OWNER: BEIJING LUYIN LIHUA PHARMACEUTICAL TECHNOLOGY DEVELOPMENT CO., LTD. Effective date: 20120809 Owner name: EMISSION AND RADIATION MEDICAL RESEARCH INSTITUTE Free format text: FORMER OWNER: RADIATION MEDICINE INST., MILITARY MEDICINE ACADEMY, P.L.A. Effective date: 20120809 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20120809 Address after: 100850 No. 27 Taiping Road, Beijing, Haidian District Patentee after: Emission and Radiation Medical Research Institute Patentee after: WUHAN GUANGGU HUMANWELL BIOLOGICAL PHARMACEUTICAL CO., LTD. Address before: 100850 No. 27 Taiping Road, Beijing, Haidian District Patentee before: Institute of Radiation Medicine of Academy of Military Medical Sciences of Chinese People's Liberation Army Patentee before: Beijing Luyin Lihua Pharmaceutical Technology Development Co., Ltd. |
|
| CX01 | Expiry of patent term | ||
| CX01 | Expiry of patent term |
Granted publication date: 20081210 |