CN101090732A - Method for treating diabetes - Google Patents

Method for treating diabetes Download PDF

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Publication number
CN101090732A
CN101090732A CNA2005800324194A CN200580032419A CN101090732A CN 101090732 A CN101090732 A CN 101090732A CN A2005800324194 A CNA2005800324194 A CN A2005800324194A CN 200580032419 A CN200580032419 A CN 200580032419A CN 101090732 A CN101090732 A CN 101090732A
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btc
cell
polypeptide
diabetes
application
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A·J·邓巴
C·戈达德
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Novozymes Biopharma AU Ltd
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Gropep Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Abstract

The present invention provides methods of treating or preventing diabetic conditions, comprising the administration of a polypeptide which has the ability to stimulate pancreatic islet ss-cell activity but which does not bind to or activate ErbB1 or ErbB4 receptors. Methods of diagnosing diabetic conditions are also provided, as are uses of the polypeptide, and compositions comprising the polypeptide.

Description

The method of treatment diabetes
The present invention relates to treatment of diabetes or prevention, be specifically related to polypeptide or corresponding nucleic molecule and treating or preventing 1 type or type 2 diabetes mellitus, perhaps postpone the application in its morbidity.
Background
With all lists of references of quoting in this description, it is for referencial use to comprise that this paper is included in any patent or patent application in.Do not admit that any list of references constitutes prior art.Author's opinion has been stated in the discussion of list of references, and the applicant keeps the accuracy of addressing inquires to citing document and right targetedly.Should clearly understand, though this paper has mentioned many prior art publications, in Australia or any other country, this list of references can not illustrate that any of these document has formed the part of this area general knowledge.
Diabetes are the human modal serious metabolic diseases of influence.It can be defined as chronic hyperglycemia, promptly owing to lack the effect of insulin relatively or definitely, sugar too much in the blood.
Diabetes are divided into two kinds of principal modes: 1 and type 2 diabetes mellitus.Type 1 diabetes is also referred to as insulin-dependent diabetes (IDDM), and it is almost all to lack relevant autoimmune disease with the pancreas beta cell that produces insulin.This beta cell disappearance causes lifelong insulin-dependent.Type 1 diabetes can betide any age, estimates that about 1% neonate can be suffered from this disease in its life.Type 2 diabetes mellitus or noninsulindependent diabetes (NIDDM) refer to be characterized as one group of disease of glucose level height (hyperglycemia) and insulin resistance in the blood, betide the impaired patient of pancreatic beta cell function.The sugar that the insulin resistance that lacks insulin and type 2 diabetes mellitus among the 1 type patient causes absorbing from blood flow reduces, so accumulates polysaccharide in the blood.These diabetes of two types all are accompanied by and shorten and important disease life expectancy, as angiopathy, blind and atherosclerosis.A kind of novel diabetes of LADA (adult latency autoimmune diabetes) have been proposed to be called, to describe the adult who diagnoses the immunology evidence (because they are anti--Gad antibody positive) that type 2 diabetes mellitus is arranged, also have type 1 diabetes.
Diabetes are one of the most general chronic diseases of developed country, are main causes of death.Except that clinical onset rate and mortality rate, the Financial cost of diabetes is huge, and only at the annual $900 hundred million that just surpasses of the U.S., expecting the popular of diabetes in 2010 will increase more than the twice, mainly be because the type 2 diabetes mellitus relevant with obesity.
The risk that diabetes take place non-Europe descendants crowd significantly increases.The foreseeable future, diabetes comprise that in the Asia South Asia can be very popular.These individualities are not having under the serious fat situation diabetes to take place, so the insulin defective may be the main component of its disease.In addition, two paradiabetess relevant (relating to malnutrition, fiber calculus pancreas diabetes and protein deficiency pancreas diabetes) have been found in India with low insulin level.
Insulin is produced by the beta cell of pancreas Langerhans' islands.Between the period of development, the islets of langerhans precursor is bred, and finally is divided into one of four kinds of main islet cells types at pancreas.Think that the formation of beta cell specifically is subjected to the adjusting of various somatomedin, cytokine and hormonal action.
The quantity of beta cell and function are very important to keeping glucose metabolism, and the serious reduction of beta cell quantity and/or function causes onset diabetes.What determine now is that beta cell quality (mass) is to keep by loss with from the balance between the precursor new life.When significantly improving (for example because fat) when insulin resistance makes insulin requirements, stimulated beta cell new life.This causes the expansion of beta cell quality, and keeps glucose metabolism.If this compensatory mechanism is impaired, for example in type 2 diabetes mellitus, just destroyed glucose metabolism.Studies have shown that in recent years, in the animal model of type 2 diabetes mellitus, beta cell new life is impaired.Therefore, stimulate beta cell new life's effectively prevent diabetes morbidity of factor, thereby represented the Therapeutic Method that comes in handy.
The main purpose of the diabetes of treatment form of ownership is identical, promptly as much as possible blood sugar level is reduced being close to normal level, thereby at utmost reduces the short-term and the long-term complications of this disease.
The long-term control of diabetes usually comprises insulinize, no matter the patient is 1 type or 2 types.In healthy individual, insulin increases the glucose of skeletal muscle picked-up and reduces the glucose that liver produces; Yet in suffering from the individuality of type 2 diabetes mellitus, insulin can not play this effect.Therefore, many type 2 diabetes mellitus patients can not produce good reaction to insulinize, even give the insulin of high dose.
The treatment of type 1 diabetes must comprise the insulin that replaces dosage, gives insulin by the outer approach of gastrointestinal tract usually.Combine with dietetic therapy and blood sugar level self-monitoring, most of 1 type patient is blood sugar control suitably.In type 2 diabetes mellitus, initial therapy is best diet program, minimizing body weight and performs physical exercise.And if when these methods no longer provide enough metabolism to control, begin pharmacotherapy.What deliver prescription usually is the medicine that promotes insulin secretion or reduce glucose level by alternate manner.Blood sugar lowering as sulphur urea or biguanide, is these patients' of open a main medicine.They stimulate insulin to produce by direct stimulation beta cell; Therefore, the effectiveness of these medicines relies on the quantity that function beta cell (beta cell quality) arranged that retains in the pancreas.
The sulphur urea is invalid in most of type 2 diabetes mellitus; In a research, the 30% new diabetics of diagnosing for the treatment of with the sulphur urea needs insulin in the first six years of treatment.Recently, it is reported to have genovariation up to 12% type 2 diabetes mellitus patient, i.e. the variation of Arg972 in the IRS-1, this variation makes them can not adopt these medicines, so they need insulinize.
Peptide growth factor has participated in various physiology and pathological process, comprises signal transduction, cell survival, differentiation, cell adhesion, cell migration, immunne response, hemoposieis, inflammation, tissue repair, atherosclerosis and cancer.In nearly all these processes, peptide growth factor interacts by the ectodomain with transmembrane receptor tyrosine kinase (RTK) and exercises its biological action.Most of RTK belongs to the little monoid of height homoreceptor, and they form in conjunction with similar part and by inductive homology of part or heterodimer and keep receptor interaction.After the inductive dimerization of part takes place, these receptor-specific tyrosine residue generation autophosphorylations in the cytoplasmic structure territory.The tyrosine residue of phosphorylation is as having SH2 or the phosphotyrosine high affine stop site in conjunction with the protein (as the p85 subunit of Shc, Grb2 and phosphoinositide 3 '-kinases (PI3-kinases)) in (PTB) territory.The activation that this causes signal transduction pathway such as mitogen-activated protein kinase approach causes complicated intracellular signal cascade reaction, the particular event that finally produces target cell.
One of RTK subfamily of the most thorough research is an ErbB family, and this family comprises four kinds of known receptor: ErbB-1 (being also referred to as EGF-R ELISA (EGFR)), ErbB-2 (being also referred to as HER2 or Neu), ErbB-3 and ErbB-4.These receptors are distributed widely in different tissues, and according to cell type and physiological condition, are used for mediating growth inhibition or inducing cell propagation and differentiation.
Many peptide growth factors are parts of ErbB receptor family.This group factor has the height sequence similarity, especially has epidermal growth factor (EGF) motif of 36-40 amino acid residue of six-cysteine.This motif contains CX 7CX 4CX 10CX 1CX 8C forms three intramolecular disulfide bond (C at interval 1-C 3, C 2-C 4, C 5-C 6), have the characteristic tricyclic structure, comprise C 1-C 3Two sulfur rings, C 2-C 4Two sulfur ring and C 5-C 6Two sulfur rings.
As if the EGF-motif that belongs to the growth factor peptides of this family all is by two exons codings that contain pinpoint interval intron, and intron is corresponding to preceding two two sulfur rings (A ring, C 1-C 3The B ring, C 2-C 4) (C encircles, C with the 3rd ring 5-C 6) border that separates.The common feature of these molecules is that they are synthetic as the bigger film precursor of striding, and strides the solubility biologically active form of film precursor through this somatomedin of Proteolytic enzyme cutting back release.
Total EGF-motif is very important for combination and activation ErbB receptor tyrosine kinase family member.Induce receptor homolog or allos dimerization, autophosphorylation and the activation of downstream signal transduction pathway subsequently in conjunction with the part of ErbB receptor, cause various physiological process, comprise cell proliferation, differentiation, migration and survival.The mammal part of ErbB family comprises EGF, transforminggrowthfactor-(TGF-α), heparin-bounding EGF-like growth factor (HB-EGF), epiregulin (epiregulin), amphiregulin, nerve-and thymus-deutero-ErbB kinase activator thing (NTAK), neuregulin (NRG) subfamily (product that comprises four kinds of genes (NRG1), NRG2, NRG3 and NRG4) and β tunicin (BTC).
Originally be purified into BTC from the conditioned medium of mice pancreatic beta cell cancer (insulinoma) cell line, it is the 32kDa glycoprotein that fibroblast, retinal pigment epithelium and smooth muscle cell is had short mitotic activity.People BTC clone is from MCF-7 MCF-7, and cattle BTC clone from bovine kidney cells is.
BTC synthesizes the precursor protein that is anchored on the film, and precursor protein can discharge the ripe somatomedin of solubility through the Proteolytic enzyme cutting.Sophisticated BTC combination also activates ErbB1 and ErbB-4 homodimer, in addition, goes back combination and activates all possible ErbB heterodimer, comprises highly carcinogenic heterodimer ErbB2-ErbB3 receptor complex.
Many researchs are emphasized, ErbB1 receptor and part TGF α specifically are that HB-EGF and BTC may be very important in pancreas growth and function.In the pancreas of whole growth, the expressed in abundance of ErbB part.Usually die from the birth ErbB1 in first week of back -/-Mice generally demonstrates pancreas epithelial cell proliferation defective, follows the island cell to postpone to be divided into the beta cell of insulin-producing, and the migration of the pancreatic islet endocrine of growing and structure form and be damaged.These defectives show that the ErbB-1 Mediated Signal Transduction is grown very important to normal beta cell.ErbB-3 -/-The pancreas of mice is grown also major defect.
Many studies have shown that, pancreas grow and function in, BTC but not EGF or TGF α can have the nonredundancy effect of uniqueness.BTC is at pancreas, and strong expression in the Langerhans' islands particularly stimulates various pancreatic cells to breed in vitro and in vivo and break up.Because the vessel cell of pancreas is expressed ErbB-1 and ErbB-4, thus think BTC can in conjunction with and activate these receptors to stimulate beta cell new life.
Quantity is big that surprising memebrane protein carries out other road montage, and the exon sequence of coding membrane spaning domain is removed in the road montage in addition, produces and the different soluble form of film combining form function.In addition the mRNA structural change that causes of road montage can be created in some tissue, different stage of development and/or difference expressed protein variant under different cell activation states.When the protein domain of the exons coding of other road montage on function to catalytic activity or combination when very important, the protein that obtains usually demonstrates difference or even antagonistic activity.
We have identified a kind of mRNA transcript of in addition road montage recently, new isotype (international patent application no PCT/AU01/00010, the GroPep Limited of its coding people BTC; Dunbar etc., 2000).This isotype is called BTC-δ 4, it lacks the 147bp of the exon 4 of coding BTC gene, cause producing the mRNA that coding has lacked the unusual BTC precursor of the C-ring of EGF domain and membrane spaning domain, and the remainder of ripe molecule comprises that A ring and B ring and " hinge " valine frame endomixis are in the terminal kytoplasm tail of the C-of truncate.Keep the hydrophobic signal sequence and lack the membrane spaning domain explanation, BTC-δ 4 may be a secretory protein.PCT/AU01/00010 proposes, and this BTC splice variant can be used for regulating the receptor-mediated activity of ErbB, therefore can be used for treating and the relevant disease of ErbB oncogene overexpression, as cancer.
We confirm then, and this splice variant is a secretory protein.Yet we find that BTC-δ 4 is combination or activation ErbB1 or ErbB4 receptor not, not in conjunction with ErbB2 or ErbB3 homodimer; Therefore our inference, BTC-δ 4 does not have functional activity, and like this for ErbB1 and ErbB4 receptor and ErbB2 and ErbB3 homodimer at least, the core EGF motif that BTC-δ 4 loses may be active necessary (Dunbar etc., 2000).
Reported the isotype HB-EGF (Loukianov etc., 1997) of the 3rd the two sulfur ring that lacks the EGF domain.In this case, the 94bp insert causes frameshit and ripe preceding the termination between exon III and the IV, produce stick signal peptide, pro-region, heparin-, and nine amino acid whose short-tails have replaced the 3rd two sulfur ring, have striden film and cytoplasm domain in conjunction with the albumen of preceding two conservative two sulfur rings of territory and EGF motif.
U.S. Patent number 6,825,159 disclose the variant of β tunicin, classify SEQ ID NO.38 as, this patent prompting, this variant has the EGF activity that complete β tunicin is active and reduce.The difference of SEQ ID NO.38 and polypeptide of the present invention is that it has comprised zero defect in all six cysteine and the C5-C6 ring.
Summary of the invention
Although BTC-δ 4 and ErbB receptor obviously can't in conjunction with, we find, BTC-δ 4 stimulates the beta cells differentiation, increase the beta cell quality, reduce the reduction of beta cell function and increase the insulin secretion of beta cell.As the beta cell differential stimulus, BTC-δ 4 is more effective than real BTC; Yet compare with real BTC, BTC-δ 4 does not have cell growth-promoting activity or this active reduction.
Aspect first, the invention provides and suffering from diabetes or be in treatment in the object of suffering from diabetes risk, prevent this disease or postpone the method for its morbidity, this method comprises that giving this object can stimulate island cell CFU-GM to be divided into the polypeptide of pancreatic beta cell, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Aspect second, the invention provides and suffering from diabetes or be in treatment in the object of suffering from diabetes risk, prevent this disease or postpone the method for its morbidity, this method comprises and gives this object a kind of nucleic acid, this nucleic acid coding can stimulate island cell CFU-GM to be divided into the polypeptide of pancreatic beta cell, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Aspect the 3rd, the invention provides the polypeptide that can stimulate island cell CFU-GM to be divided into pancreatic beta cell treats in diabetics, prevents this disease or postpones the application of its morbidity, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Aspect the 4th, the nucleic acid that the invention provides coding can stimulate island cell CFU-GM to be divided into the polypeptide of pancreatic beta cell is suffering from diabetes or is being in treatment in the object of suffering from diabetes risk, prevents this disease or postpones the application of its morbidity, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Aspect the 5th, the invention provides the polypeptide that can stimulate island cell CFU-GM to be divided into pancreatic beta cell can suffer from diabetes or be in treatment in the object of suffering from diabetes risk, prevent this disease or postpone application in the medication preparation of its morbidity, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Aspect the 6th, the nucleic acid that the invention provides coding can stimulate island cell CFU-GM to be divided into the polypeptide of pancreatic beta cell can suffer from diabetes or be in treatment in the object of suffering from diabetes risk, prevents this disease or postpone application in the medication preparation of its morbidity, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Preferably, compare with real BTC, the ErbB1 of this polypeptide activation object and the ability of ErbB4 receptor significantly reduce, and stimulate island cell CFU-GM not stimulate the propagation of other cell type to the pancreatic beta cell differentiation phase.Preferably, compare with real BTC, this polypeptide does not have fibroblast growth promoting activity or should activity reduce.Preferably, compare with real BTC, this polypeptide does not have epithelial cell growth to promote active or should activity reduce.
Preferably, described diabetes are type 2 diabetes mellitus or type 1 diabetes.
Aspect the 7th, the invention provides treatment, prevent diabetes or postpone its morbidity compositions, said composition comprises the polypeptide and the pharmaceutically acceptable carrier that can stimulate island cell CFU-GM to be divided into pancreatic beta cell, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Aspect the 8th, the invention provides treatment, prevent diabetes or postpone its morbidity compositions, said composition comprises coding can stimulate island cell CFU-GM to be divided into the nucleic acid and the pharmaceutically acceptable carrier of the polypeptide of pancreatic beta cell, compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
Preferably, compare with real BTC, this polypeptide does not have the ability of irritation cell type propagation or this ability to reduce, and compares with real BTC, and ability or this ability that this polypeptide does not activate ErbB1 or ErbB4 receptor reduce.Preferably, compare with real BTC, this polypeptide does not have fibroblast growth promoting activity or should activity reduce.Preferably, compare with real BTC, this polypeptide does not have epithelial cell growth to promote active or should activity reduce.
Preferably, compare with real BTC, ability or this ability that this polypeptide does not activate ErbB2 or ErbB3 homodimer reduce.
As the side effect of treatment, this polypeptide makes the risk of object generation cancer may be lower than real BTC and handles the risk that produces.
Preferably, the basic homology of member of this polypeptide and EGF family.More preferably, this polypeptide and epidermal growth factor, transforminggrowthfactor-, heparin-bounding EGF like growth factor, epiregulin, amphiregulin, nerve-and basic homology of thymus-deutero-ErbB kinase activator thing (NTAK), neuregulin subfamily member (NRG1, NRG2, NRG3 and NRG4) or β tunicin (BTC).More preferably, this polypeptide and BTC-δ 4 basic homologies.Most preferably, this polypeptide is BTC-δ 4.BTC-δ 4 amino acid sequence of polypeptide are shown in Fig. 4 (SEQ ID NO:11-15).
This polypeptide can be:
(a) splice variant of BTC does not wherein have C 5-C 6Two sulfur rings, and have this two sulfur ring in the gene of the real BTC that encodes usually; Or
(b) sequence and (a) the homologous substantially polypeptide of molecule, or
(c) (a) analog of molecule, fragment, mutant, derivant or allele variant.
This polypeptide can be:
(a) splice variant of BTC does not wherein have C 5-C 6Two sulfur rings, this two sulfur ring is usually by the nucleic acid sequence encoding that exists in the gene of the real BTC of coding, the encode remainder of nucleotide sequence of real BTC peptide molecule comprises A ring and B ring and " hinge " valine frame endomixis nucleotide sequence in the C-of coding truncate end kytoplasm tail;
(b) sequence and (a) the homologous substantially polypeptide of molecule; Or
(c) fragment (a), analog, variant or derivant.
Preferably, this polypeptide can be used as the beta cell differentiation factor, increases the beta cell quality, reduce the reduction of beta cell function and/or the insulin secretion of increase beta cell.
Described fragment, analog, variant or derivant can have following feature:
(a) one or more amino acid residues are by conservative or non-conservative amino acid residues, and preferred conservative amino acid residues replaces; The amino acid residue of described replacement can the yes or no genetic code residue of coding;
(b) one or more amino acid residues comprise substituent group;
(c) mature polypeptide is blended in another kind of chemical compound, to increase the half-life of this polypeptide; Or
(d) be blended in other aminoacid of mature polypeptide.
As the side effect of treatment, this polypeptide makes the risk of object generation cancer may be lower than the risk that the BTC treatment produces.
Preferably, object has in the following diabetes risk factor one or multinomial: the glucose tolerance is impaired, metabolism syndrome, 1 type or type 2 diabetes mellitus family history, obesity, polycystic ovarian syndrome, hypertension or hypercholesterolemia.
Should clearly understand, the variant form that comprises other member of the EGF family that uses peptide growth factor in the scope of the present invention, it can be used as external and/or intravital beta cell differentiation factor, increases the beta cell quality in external and/or body, reduce the reduction of beta cell function and/or increase the beta cell insulin secretion in external and/or body, and this paper is referred to as " similarly growth factor variants ".In these variant forms, be present in the amino acid residue disappearance in the real polypeptide usually, its disappearance mode is similar to the disappearance among the BTC-δ 4.Preferably, be not present in C in the real molecule usually 5-C 6Two sulfur rings are similar to the disappearance among the BTC-δ 4.The suitable real polypeptide that is selected from EGF family comprises epidermal growth factor, transforminggrowthfactor-, heparin-bounding EGF like growth factor, epiregulin, amphiregulin, nerve-and thymus-deutero-ErbB kinase activator thing (NTAK) and neuregulin subfamily member (NRG1, NRG2, NRG3 and NRG4).The model of action of estimating these variant forms is similar to BTC-δ 4 polypeptide of the present invention, and condition is that they have the above-mentioned functions feature.
In case should be understood that and known amino acid sequence of polypeptide described herein, just can be synthetic or the similar variant of the synthetic BTC-δ 4 of recombination method and other somatomedin by chemical method well known in the art such as solid-phase polypeptide.
Preferably, this peptide molecule has the aminoacid sequence of human origin.Yet the present invention also comprises the BTC-δ 4 that uses non-human mammal; BTC-δ 4 polynucleotide of the present invention are not difficult as the probe that separates the corresponding polynucleotide of other mammalian cell with this area conventional method.
Therefore, polypeptide of the present invention can be recombinant polypeptide, isolating natural generation polypeptide or synthetic polypeptide, preferred recombinant polypeptide.
The inventive method can be used for treating type 2 diabetes mellitus, and prevents type 2 diabetes mellitus or postpone its morbidity.And because the feature of type 1 diabetes is the disappearance of β cell and completely losing of β cell function, the present invention also can be used for treating type 1 diabetes, perhaps prevents type 1 diabetes or postpones its morbidity.
Randomly, polypeptide of the present invention can be with the immunosuppressant administration.Suitable immunosuppressant comprises the sharp former times monoclonal antibody (Remicade) of corticosteroid, TNF-Alpha antibodies such as English and soluble TNF-α receptor such as Yi Tanxipu (Enbrel).Can preferred this conjoint therapy in type 1 diabetes or LADA (any new β cell still is subjected to immune attack, unless suppress immune attack).
Can imagine that compare with adopting real BTC, the inventive method reduces carcinogenic risk.We find that BTC-δ 4 can not activate ErbB1 and ErbB4 receptor, have therefore prevented the ErbB-stimulated cell proliferation, thereby reduce or avoided becoming the risk of cancer of treatment side effect.
Consider especially, the inventive method and compositions are suitable for the therapeutic treatment of human diabetes, they also can be used for veterinary treatment, comprise treatment fellow creature such as dog and cat, and tame pack animal, cattle and sheep, or zoo animal such as inhuman Primate, felid, Canis animals, bovid and ungulate.
Usually the form with pharmaceutical composition gives polypeptide of the present invention.The method of pharmaceutical compositions and medicine carrier are well known in the art, as " Lei Mingdun pharmaceutical science " (Remington ' s Pharmaceutical Sciences), the 20th edition, Williams and Wilkins, textbooks such as Pennsylvania, America are described.
Carrier or diluent and other excipient will depend on route of administration, and those skilled in the art can determine only dosage form for each concrete condition.
Can give The compounds of this invention and compositions by any suitable pathways, those skilled in the art can determine only approach and dosage for the disease of being treated.Dosage determined by the doctor in charge or veterinary, depend on age of sanatory characteristic and state, institute's treatment target and health status, route of administration and the treatment formerly that may give.
Brief Description Of Drawings
Figure 1A has shown with real people BTC and splice variant people BTC-δ 4 in RT-PCR detection MCF-7 cell (swimming lane 1) and the human breast skin flbroblast (swimming lane 2).Swimming lane 3 is contrasts of no cDNA template.Figure 1B shows with comprising coding people BTC aminoacid D 32-Y 111The corresponding Southern trace that carries out of the cDNA probe of polynucleotide.
Fig. 2 A has compared the part nucleotide of real people BTC and splice variant people BTC-δ 4 and the aminoacid sequence of inferring (with an alphabetical aminoacid coded representation).To go into pBluescript II SK available from the RT-PCR product cloning of MCF-7 cDNA among Figure 1A, and the order-checking insert.Shown 147bp disappearance point (representing) sequence on every side with arrow down.Fig. 2 B has shown the complete nucleotide sequence of BTC-δ 4 cDNA and the aminoacid sequence of inferring.
Fig. 3 has compared the complete amino acid sequence of real people BTC and splice variant people BTC-δ 4 in vacation is intended comparing.Horizontal dotted line in BTC-δ 4 sequences is represented to compare with real hBTC sequence and is lost amino acid whose site.Following sketch map has shown the global structure of comparing real people BTC with BTC-δ 4.
Fig. 4 has shown BTC-δ 4 amino acid sequence of polypeptide (BTC-δ 41-129, BTC-δ 41-94, BTC-δ 4 32-94, BTC-δ 4 32-129, BTC-δ 4 32-111With BTC-δ 4 95-129).
Fig. 5 explanation is used for the construction of the recombinant expressed and purification of BTC and BTC-δ 4.Fig. 5 A is the sketch map that is respectively applied for the construction of BTC and BTC-δ 4.Fig. 5 B has shown and has used C 4The analysis RP-HPLC of post estimates the result of each BTC or BTC-δ 4 preparation purity.
It is secretory proteins that Fig. 6 demonstrates BTC-δ 4.A: with BTC-FLAG, BTC-δ 4-FLAG or empty carrier (pcDNA3.1) transient transfection COS-7 cell.After the transfection 72 hours, collect culture medium and prepare cell lysate.Western engram analysis culture medium and cell lysate with anti--FLAG M2 antibody.B: transfection COS-7 cell as mentioned above, with the elisa assay culture medium or the impermeability fixed cell of anti--FLAG M2 antibody.It is noted that with anti--BTC extracellular domain antibody (R﹠amp; D Systems) also can obtain similar Western trace and ELISA result's (data not shown).
Fig. 7 demonstrates BTC but not BTC-δ 4 has stimulated Balb/c 3T3 fibroblast proliferation, also demonstrate BTC but not BTC-δ 4 in radioreceptor test in conjunction with ErbB1 and ErbB4.BTC or the BTC-δ 4 (A) that Balb/c 3T3 fibroblast increases with independent concentration or in the presence of the BTC of fixed concentration, hatch with the BTC-δ 4 (B) of concentration increase.In the presence of BTC or BTC-δ 4 that concentration increases [ 125I]-BTC of labelling and express the AG2804 cell (C, D) of ErbB1 or competitive combination of Chinese hamster ovary celI (E, F) of ErbB4 transfection.
Fig. 8 demonstrates BTC but not BTC-δ 4 has induced ErbB1 and the ErbB4 receptor tyrosine phosphorylation in AG2804 and the CHO-ErbB4 cell respectively.Be untreated (-) or handle the lysate of (10nmol/l BTC, 10nmol/l BTC-δ 4 or 10nmol/l BTC+10nmol/l BTC-δ 4).Make cellular immunization precipitation (IP) with anti--ErbB1 (AG2804) or anti--ErbB4 (CHO-ErbB4) antibody, carry out trace and detection with anti--phosphorylated tyrosine antibody (α PY) then.Also the elution trace also detects with anti--ErbB1 or anti--ErbB4 antibody again.
Fig. 9 demonstrates BTC and BTC-δ 4 induces the AR42J cell differentiation.The AR42J-B20 cell was hatched 48 hours with 2 nM activin A and 1nM BTC (figure A and C) or BTC-δ 4 (figure B and D), and is fixing then and dye with anti-insulin-antibody (arrow " 1 ") and DAPI (arrow " 2 "), and DAPI dyes nuclear.In the cell that activin A and BTC-δ 4 handles, many cellular expression insulins, but nuclear is by fragmentation (arrow " 3 ") or lose.Amplification, figure A and B: * 100; Figure C and D: * 400.
Figure 10 demonstrates BTC but not BTC-δ 4 has improved activin-inductive AR42J apoptosis.Handled the AR42J-B20 cell 48 hours with 2nM activin A and 1nM BTC (A) or BTC-δ 4 (B).Represent the TUNEL-positive cell with arrow.
Figure 11 demonstrates BTC-δ 4 is given the rat reduction plasma glucose concentration of STZ-processing and improves the glucose tolerance.A: at the 0th day (new born animal of an age in days) injection STZ (85 μ g/g), injected BTC or BTC-δ 4 (3pmol/g) since the 0th day every day, and measure plasma glucose concentration, amount to 5 days.Value is meansigma methods ± S.E. zero, and STZ organizes (n=5); ●, 4 groups of STZ/BTC-δ (n=5); △, STZ/BTC organizes (n=5); *P＜0.05 to the STZ group.B: on the rat at 2 monthly ages, carry out glucose tolerance test.Left figure, the change of plasma glucose concentration.Right figure, the change of plasma insulin concentration.Value is meansigma methods ± SE.Zero, STZ organizes (n=5); ●, 4 groups of STZ/BTC-δ (n=5); △, STZ/BTC organizes (n=5), (---): normal SD rats (n=3). *To P＜0.05 of STZ group, *P＜0.01 to the STZ group.
The positive vessel cell (A) of the PDX-1 that measured in the 4th day and island like cell bunch (ICC) quantity (B) that Figure 12 demonstrates in the described research of Figure 11.(A): positive vessel cell (arrow " 1 "): the PDX-1 (arrow " 2 ") of PDX-1-: cytokeratin.(B): ICC (arrow " 3 "): cytokeratin (arrow " 4 "): insulin (arrow " 5 "): DAPI.STZ: with the rat of saline treatment STZ-injection. *P＜0.05 to STZ. *P＜0.01 to STZ.Amplification, A: * 400; B: * 100.
Detailed Description Of The Invention
Except as otherwise noted, all scientific and technical terminologies used herein are identical with the implication that one skilled in the art of the present invention understand. Although can adopt to this paper under the similar or equivalent any materials and methods of content implement or test the present invention, preferred materials and methods has been described.
Definition
Singulative used herein " one ", " a kind of " and " this " comprise corresponding plural implication, are not like this unless explicitly point out in the literary composition. Therefore, for example, mention " a kind of enzyme " and comprise multiple such enzyme, mention " seed amino acid " and refer to one or more amino acid.
In specification of the present invention and appended claims, except because representation language or must in the implication literary composition requirement be arranged in addition, term " comprises " and is used for the description of comprising property, and there is not or has added further feature in feature under namely explanation exists but do not get rid of in the various embodiments of the present invention.
Term used herein " object " refers to suffer from need to be with any animal of the diabetes of pharmaceutically active substances treatment. Object can be the people, perhaps can be domestic animal or fellow creature. Be suitable for human therapeutic treatment although specifically considered the compounds of this invention, but it also can be used for veterinary treatment, comprise treatment fellow creature such as dog and cat, and family's pack animal, ox and sheep, or zoo animal such as inhuman Primate, cats, canid, bovid and ungulate.
Term used herein " treatment effective dose " refers to effectively to produce required therapeutic response, is easy to the consumption of the compounds of this invention of the disease by giving pharmaceutically active substances treatment such as prevention or treatment.
Certainly, concrete " treatment effective dose " because various factors changes, as the health of the disease specific for the treatment of, object and clinical history, type, treatment time, the characteristic of concurrent treatment (if there is) and the structure of used concrete preparation and compound or derivatives thereof for the treatment of animal.
Peptide concentration in the therapeutic combination is inessential, but should be the content that can effectively treat diabetes or prevention or postpone its morbidity. In all methods that adopt BTC-δ 4 polypeptide, term used herein " effective dose " refers to be enough to cause with 95% confidence level the amount of significance,statistical reaction (namely only because the p of the effect that probability causes＜0.05). Can pass through experience, according to cell in vitro to this polypeptide or contain the reaction of preparation of this polypeptide and animal used as test is measured the polypeptide consumption to this polypeptide or the reaction that contains the preparation of this polypeptide. The polypeptide consumption should be enough to cause in vivo beta cell insulin secretion increase in beta cell differentiation, the interior beta cell mass penalty of body, body interior Instreptozotocin Induced decline minimizing and/or the body. On average, the patient's of the about 75kg of body weight daily dose is at least 0.001mg/kg, preferred 0.01mg/kg, and to about 20mg/kg, preferred 1mg/kg body weight.
" medicine carrier " used herein is for the pharmaceutically acceptable solvent, suspending agent, excipient or the carrier that BTC-δ 4 and/or other pharmaceutically active substances are delivered to object. Carrier can be liquid or solid, selects according to the plan administering mode.
Term used herein " diabetes " comprises type 1 diabetes and diabetes B, and type 1 diabetes is also referred to as insulin-dependent diabetes mellitus (IDMM), and diabetes B is also referred to as adult-onset diabetes (NIDDM).
Splice variant of the present invention is called BTC-δ 4, and will the encode polynucleotides of BTC-δ 4 of this paper are called BTC-δ 4 polynucleotides. Used term " real " refers to 178 amino acid whose total lengths or ripe solubility BTC albumen when mentioning BTC.
Term " sequence of basic homology " refers to be equivalent on the function polypeptide of particular B TC-δ 4 sequences as herein described, comprises replacement, disappearance or insertion in the disclosed especially peptide sequence.
Term when mentioning polypeptide of the present invention " fragment ", " analog ", " variant " and " derivative " have referred to keep the therewith essentially identical biological function of polypeptide or active molecule. Therefore, analog comprises front albumen (proprotein), and it can produce activated mature polypeptide and activate by cutting front protein part.
Term " fragment " should clearly be interpreted as part or the domain of the arbitrary size that comprises full length sequence, as long as this fragment has functional activity. Term " fragment of sequence " or " part of sequence " refer to the truncated sequence of described original series. The length of truncated sequence (nucleic acid or peptide sequence) can be different, and minimum dimension is for being enough to provide the size that has at least the function suitable with described original series and/or active sequence, and full-size is unimportant. In some embodiments, full-size is not more than required activity and/or the required size of function that original series is provided usually basically. The length of the amino acid sequence of brachymemma generally is at least about 5 amino acid. Yet more generally, the length of this sequence is at least about 50 amino acid, preferably is at least about 60,80,100,120,150,200 or 220 amino acid. But other amino acid of fragment sequence side joint, or can modify according to those skilled in the art's known method. The protein of modifying must keep at least a BA of native protein. This fragment can be used for oxidation or reduction form, perhaps with coupling or blocking group coupling. For example, they can be coupled to the carrier molecule.
Protein " variant " comprises that the homologue and containing of albumen shown in Figure 4 can make the protein of the conservative replacement that this albumen secondary structure remains unchanged. The example of this conservative replacement comprises hydrophobicity, size and electric charge and the original essentially identical amino acid residue of amino acid residue. This replacement is protein chemistry field known by the technical staff normally. For example, conservative replacement comprises the proline substituted glycinic acid, and vice versa; Alanine or valine substituted glycinic acid, vice versa; Isoleucine replaces leucine, and vice versa; Histidine replaces lysine, and vice versa; Serine replaces asparagine, and vice versa; Threonine replaces cysteine, and vice versa; Serine or alanine replace threonine, and vice versa; Glutamine replaces asparagine, and vice versa; Tryptophan replaces tyrosine, and vice versa; Arginine replaces glutamic acid, and vice versa.
Term protein " derivative " comprises the protein that has replaced one or several amino acid residues with 20 kinds of amino acid whose natural generations of natural generation or synthetic amino acid homologue. The example of this homologue is 4-hydroxy-proline, 5-oxylysine, 3-Methyl histidine, homoserine, Beta-alanine, 4-Aminobutanoicacid, ornithine, nor-leucine, norvaline, hydroxy-proline, thyroxine, GABA, homoserine, citrulling etc.
Natural amino acid residue used herein is 20 seed amino acid residues (being alanine, aspartic acid, asparagine, arginine, cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine) common in the protein, and comprises these amino acid whose D shapes and L shaped.
Synthesizing amino acid residue used herein is included in the D shape of any other amino acid residue that produces that find in the situation that find in the protein, natural or synthetic and L shaped. The synthesizing amino acid residue includes but not limited to: Beta-alanine, ornithine, nor-leucine, norvaline, hydroxy-proline, thyroxine, GABA, homoserine, citrulling etc.
When the scope of expression value, should clearly understand, this scope comprises the bound of this scope and all values between the bound.
Should clearly understand, the invention is not restricted to concrete materials and methods as herein described, because they can change. Should be understood that also it only is to describe the specific embodiment that this paper uses the purpose of term, be not intended to limit the scope of the invention, the scope of the invention only is subjected to the restriction of appended claims.
Except as otherwise noted, the present invention adopts chemistry, protein chemistry, molecular biology and the zymetology technology of the routine that those skilled in the art can use. These technology are that this area staff knows, and detailed explanation is arranged in the literature. Referring to for example, Coligan, Dunn, Ploegh, Speicher and Wingfield: " prior art in the protein science " (Current protocols in Protein Science) (1999) I volume and II roll up (John Wiley and Sons Inc.); Sambrook, Fritsch and Maniatis: " molecular cloning: laboratory manual " (Molecular Cloning:A Laboratory Manual) (2001); Shuler, M.L.: " bioprocess engineering: basic conception " (Bioprocess Engineering:Basic Concepts) (second edition, Prentice-Hall International, 1991); Glazer, A.N., DeLange, R.J. and Sigman, D.S.: " chemical modification of protein " (Chemical Modification of Proteins) (North Holland Publishing Company, Amsterdam, 1975); Graves, D.J., Martin, B.L. and Wang, J.H.: " common translation and the posttranslational modification of protein: the principles of chemistry and biological effect " (Co-and post-translational modification of proteins:chemical principles and biological effects) (Oxford University Press, 1994) Lundblad, R.L. (1995) " protein modification technology " (Techniques in protein modification) .CRC Press, Inc. Fla. Berkeley village; And Goding, J.W " monoclonal antibody: principle and put into practice " (Monoclonal antibodies:principles and practice) (Academic Press, New York: the third edition. 1996).
The inventive method can comprise
(a) before giving the second pharmaceutically active substances, give BTC-δ 4 together or afterwards, with the treatment diabetes; Or
(b) unite and give BTC-δ 4 and the second pharmaceutically active substances, with the treatment diabetes.
Abbreviation used herein is as follows:
The bp base-pair
BTC β tunicin
The CM culture medium
DMEM Dulbecco basal essential medium
The EGF EGF
EIA enzyme immunity test
The ELISA enzyme-linked immunosorbent assay
The IDMM IDD
The IGT glucose-tolerant is impaired
LADA adult's latency autoimmune diabetes
The NIDMM adult-onset diabetes
The PCR polymerase chain reaction
The PP pancreatic polypeptide
PBS phosphate-BS
The RT-PCR reverse transcriptase PCR
The SD standard deviation
The SDS-PAGE SDS-PAGE
The STZ streptozotocin
Prediabetes is also referred to as glucose tolerance impaired (IGT), affects about 41,000,000 Americans, and it is type 2 diabetes mellitus and cardiovascular diseases's a omen.ADA (ADA) is defined as blood sugar level with prediabetes and is higher than normal value, but not high disease to the level that is diagnosed as type 2 diabetes mellitus.Except reducing body weight and increase exercise activity, just carrying out clinical trial and detecting in the prediabetes progress to containing the effect of the nutritional supplement of chromium picolinate before the chronic disease.
May cause diabetes or may the basic disease relevant comprise metabolism syndrome (hypertension with diabetic syndrome, obesity/overweight and hypercholesterolemia), hemochromatosis, chronic pancreatitis, polycystic ovarian syndrome (PCOS), carcinoid syndrome, pancreatic surgery or wound, the hypophysis hyperactivity, adrenal gland's hyperactivity, pancreatic insufficiency, acromegaly, cushing's syndrome, cystic fibrosis, adenocarcinoma, somatostatinoma, aldosteronoma-inductive hypokalemia, pheochromocytoma, primary aldosteronism, Wal Fu Lamu syndrome, leprechaunism, the Rabson-Mendenhall syndrome, the stiff man syndrome, autoimmunity lymphocytic hyperplasia syndrome; Hyperprolactinemia; Hyperthyroidism; POEMS or Crow-Fukase syndrome (polyneuropathy, organomegaly, endocrinopathy, M protein and skin change); Prolactinoma; The Kearns-Sayre syndrome; Excessive and the 2 type multiple endocrine deficiency syndromes of nicotinic acid.
Specifically, the risk factor of type 2 diabetes mellitus comprise one or more IGT, type 2 diabetes mellitus family history, obesity, hypertension, hypercholesterolemia and sedentary lifestyle.Known some race specifically is that the sickness rate of type 2 diabetes mellitus among African, Spaniard and chicano, Australian aboriginal and the pacific island state resident is higher.Also comprise pregnant correlative factor, because gestational diabetes mellitus, miscarriage, stillbirth are arranged before or the big baby or have the baby's of birth defect medical history all can raise of giving a birth with the type 2 diabetes mellitus sickness rate.
Recently report, compare as if the gene mutation that is called little ubiquitin dependency trim (SUMO-4) among the type 1 diabetes patient is more frequent, this gene relevant (Guo etc., 2004 with type 1 diabetes in some families with the individuality that does not have disease; Bohren etc., 2004).
The method of well known diagnosing diabetes.General diabetes with two kinds of testing and diagnosing people:
(1) blood sugar level of fasting: " golden standard " of diagnosing diabetes is overnight fasting (late night to morning is dietary intake not) back blood sugar level height.It is diabetes that the value of at least twice mensuration is higher than 140mg/dl then diagnosable.The fasting sugar level of normal subjects is 70-110mg/dl.
Oral glucose tolerance test (OGTT): can carry out the oral glucose tolerance test in doctor's office or laboratory.Stimulate 5 blood sugar levels of mensuration in back 3 hours at glucose.Object begins test at fasting state (do not have other food or beverage to take at least 10 hours outside dewatering, but no longer than 16 hours).Get initial sample of blood, give beverage (the 75 gram glucoses of the high glucose of object then; The gravid woman is 100 grams).After drinking high glucose beverage, got blood sample then in 30 minutes, 1 hour, 2 hours and 3 hours again.In the non-diabetic people, glucose stimulates the glucose level in the blood of back to raise, but gets back to normal level thereafter rapidly.In diabetics, glucose stimulates the back glucose level to be elevated to more than the normal value, be returned to normal level then will be slowly many.
The mammal part of ErbB family comprises EGF (Savage etc., 1972), transforminggrowthfactor-(TGF-α) (Marquardt etc., 1984), heparin-bounding EGF like growth factor (HB-EGF) (Higashiyama etc., 1991), epiregulin (Toyoda etc., 1995), amphiregulin (Shoyab etc., 1989), neural-and thymus-deutero-ErbB kinase activator thing (NTAK) (Higashiyama etc., 1997), neuregulin (NRG) subfamily and β tunicin (BTC) (Shing etc., 1993), wherein neuregulin (NRG) subfamily comprises the product of four kinds of genes: (NRG1) (Marchionni etc., 1993), NRG2 (Chang etc., 1997; Carraway etc., 1997), NRG3 (Zhang etc., 1997) and NRG4 (Harari etc., 1999).
Always as described in the PCT/AU01/00010, it is for referencial use to include its full content in this paper for BTC-δ 4 polypeptide and its variant.This area can extensively obtain to produce the appropriate method of polypeptide.The preferred recombinant DNA technology that adopts.
For example, BTC-δ 4 polynucleotide sequences separate from the MCF-7 cell.It contains the open reading frame of 129 the amino acid whose polypeptide of encoding.This polynucleotide sequence is identical with people BTC, except having lacked 147bp (49 aminoacid of encoding) in the open reading frame, causes lacking C 5-C 6Two sulfur rings, this two sulfur ring is present in (referring to Fig. 2 B and 3) in the EGF domain usually.Its generation is the result of other the road montage (exon is skipped) of mRNA of one of people BTC gene extron.
BTC-δ 4 polynucleotide can be available from the various cells source of the mRNA that expresses BTC-δ 4 codings.The inventor has identified many suitable people's cell source of BTC-δ 4 polynucleotide, includes but not limited to: kidney, liver, pancreas and various breast cancer cell line such as MCF-7.
For example, the polynucleotide of coding BTC-δ 4 polypeptide can be available from the cDNA from separate in the cell source and purified RNA is cloned.Available technology preparation clone's well known in the art cDNA library, the available and complementary substantially nucleotide probe of BTC gene any part screens BTC-δ 4 coded DNA in this library.Also can utilize various PCR clone technologies to obtain BTC-δ 4 polynucleotide of the present invention.
Therefore, can utilize oligonucleotide primers to obtain the polynucleotide that code book is invented BTC-δ 4 polypeptide with PCR, described primer comprises the polynucleotide sequence of a coding BTC gene part.This primer preferably comprises 5 of least significant end ' and 3 ' coding region.This oligonucleotide primers more preferably has following sequence, or with the homologous substantially sequence of following sequence:
5′GAGCGGGGTTGATGGACCGG 3′(SEQ ID NO：1)
5′TTAAGCAATATTTGTCTCTTC 3′(SEQ ID NO：2)
Use carrier of the present invention, transform or transfection host cell as cloning vehicle or expression vector.Those skilled in the art can utilize various expression vector/host systems to come recombinant expressed BTC-δ 4 polypeptide equally well.This system includes but not limited to: the antibacterial of microorganism as transforming with the recombinant phage dna that comprises required BTC-δ 4 polynucleotide encoding sequences, plasmid DNA or cosmid DNA expression vector; Yeast with the recombinant yeast expression vector conversion that comprises required BTC-δ 4 polynucleotide encoding sequences; Insect cell system with recombinant virus expression vector (as the baculovirus) transfection that comprises required BTC-δ 4 polynucleotide encoding sequences; Or with the zooblast system of the suitable mammalian expression vector transfection that comprises required BTC-δ 4 polynucleotide encoding sequences.
Can in all sorts of ways suitable DNA sequence is inserted carrier.Usually, with method well known to those skilled in the art DNA sequence is inserted suitable restriction endonuclease site.
The DNA sequence operability of inserting expression vector is connected in expression control sequenc (promoter), and is synthetic to instruct mRNA.According to used host/vector system, can adopt any suitable transcribing/translate element.For example, during with prokaryotic cell such as escherichia coli (E.coli) clone, can adopt trc or T7 promoter; When cloning, can adopt separation from the genomic promoter of mammalian cell (as the mice metallothionein promoter) or separate the promoter (as early stage immediately (CMV) promoter of human cytomegalic inclusion disease virus) of the virus in these cells, grow with mammalian expression systems.The promoter transcription insertion sequence that also can utilize recombinant DNA or synthetic technology to produce.Expression vector also contains ribosome binding site and the transcription terminator that is useful on the startup translation.Also need the specificity enabling signal effectively to translate the coded sequence that inserts.These signals comprise that ATG starts codon and flanking sequence.In that whole BTC-δ 4 coded sequences (the startup codon and the flanking sequence that comprise himself) are inserted under the situation of suitable expression vector, do not need extra translation control signal.Yet, under the situation of only inserting a part of BTC-δ 4 coded sequences, must provide exogenous translation control signal, comprise that ATG starts codon.
And, start codon must with the reading frame homophase of BTC-δ 4 coded sequences, to guarantee the translation of whole insert.These allos translation control signals and startup codon can be various sources, comprise natural and synthetic source.Can improve expression efficiency by comprising attenuation sequence, enhancer element etc.
In addition, but expression vector preferably comprises one or more selectable marker genes, to be provided for selecting transforming or the phenotypic characteristic of the host cell of transfection, and as eukaryotic neomycin (G418) resistance, or prokaryotic cell such as colibacillary amicillin resistance.
The available carrier that contains dna molecular of the present invention, and suitable promoter or control sequence conversion or transfection suitable hosts make this albumen of this host expresses.
The representative example of suitable host includes but not limited to: bacterial cell, as escherichia coli; Insect cell such as fruit bat (Drosophila) and Sf9; And zooblast such as CHO, COS or 293 cells.
Can produce the gene outcome of recombination sequence coding in a usual manner with the construction in the host cell.Perhaps, the peptide synthesizer of available routine produces polypeptide of the present invention with synthetic method.
Can in all sorts of ways from the reconstitution cell culture and to reclaim and the polypeptide of the present invention of the above-mentioned various different carriers of purification/host expression system generation, these methods comprise ammonium sulfate or ethanol precipitation, sour extracting, anion or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatograph, affinity chromatography, hydroxylapatite chromatography and reversed-phase high-performance liquid chromatography (HPLC).If desired, in finishing required polypeptide configuration, can adopt the protein refolding step.
This polypeptide can be the product of purified product, separation self-organizing or cell source, chemosynthesis step or the product that produces with protokaryon or eucaryon host by recombinant technique.Produce used host in the step according to reorganization, polypeptide of the present invention can be glycosylation or nonglycosylated.
Can obtain that those skilled in the art can be identified and be used for the method for the homologous substantially polypeptide of analog, fragment, mutant, derivant or allele variant of of the present invention, sequence and BTC-δ 4 or BTC-δ 4, for example, is the ability of differentiation by estimating this polypeptide at external evoked pancreatic cell, or estimates the ability of the diabetic symptom order of severity that reduces diabetes animal model.
The analog, fragment, mutant, derivant or the allele variant that are used for BTC-δ 4 of the present invention preferably have following extracorporeal biology characteristic:
When (1) adopting the described method of embodiment 5, this polypeptide does not replace radiolabeled ripe BTC on the ErbB1 of people's lung fibroblast (AG2804) or the ErbB4 receptor.
When (2) adopting the described method of embodiment 5, this polypeptide is not induced the ErbB1 of people's lung fibroblast (AG2804) or the tyrosine phosphorylation of ErbB4 receptor.
When (3) adopting the described method of embodiment 6, this polypeptide stimulates the AR42J-B20 cell differentiation just as real BTC with activin A, but compares with real ripe BTC, and this polypeptide promotes that the ability of cell survival is very different.
The available dosage unit forms that contains conventional nontoxic pharmaceutically acceptable carrier, adjuvant and carrier with oral, rectum, gastrointestinal tract is outer or inhalation route gives The compounds of this invention.That term gastrointestinal tract used herein comprises outward is subcutaneous, intravenous, intramuscular, intrathoracic, intracranial, injection or infusion techniques.
The present invention also provides suitable part, oral, aerosol and the outer pharmaceutical dosage form of gastrointestinal tract, is used for the new method of the present invention's treatment.The The compounds of this invention of orally give can be tablet, aqueous or oiliness suspending agent, lozenge, tablet, powder agent, granule, Emulsion, capsule, syrup or elixir.Composition for oral administration can contain one or more materials that are selected from down group: sweeting agent, flavoring agent, coloring agent and antiseptic, purpose are to produce pharmaceutically exquisite and delicious preparation.Tablet contains active component, is mixed with nontoxic pharmaceutically acceptable excipient, and this excipient is applicable to the production tablet.
These excipient can be (for example): inert diluent, as calcium carbonate, lactose, calcium phosphate or sodium phosphate; Granulating agent and disintegrating agent are as corn starch or alginic acid; Binding agent is as starch, gelatin or Radix Acaciae senegalis; Or lubricant, as magnesium stearate, stearic acid or Pulvis Talci.Tablet can be a coating not, perhaps can be with the known technology coating, postponing disintegrate and the absorption in gastrointestinal tract, thereby provides slow releasing function in a long time.For example, can adopt time-delay material such as glyceryl monostearate or distearin.Also available U.S. Patent number 4,256,108; 4,160,452 and 4,265,874 described technology are carried out coating, ooze the treatment tablet with the grade that is formed for controlled release.
Can be by BTC-δ 4 and the pharmaceutically active substances injected or progressively perfusion is used for the inventive method separately or together outside gastrointestinal tract.Administration can be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, intracavity or transdermal administration.In in vitro study, medicine can be added or is dissolved in suitable biology of acceptable buffer and add cell or tissue.
The preparation of gastrointestinal tract external administration comprises sterile aqueous or non-aqueous solution, suspension and Emulsion.The example of non-aqueous solvent is propylene glycol, Polyethylene Glycol, vegetable oil such as olive oil and injectable organic ester such as ethyl oleate.The aqueous carrier comprises water, alcoholic solution/aqueous solution, Emulsion or suspension, comprises saline and buffering culture medium.The outer carrier of gastrointestinal tract comprises sodium chloride solution, woods Ge Shi dextrose, dextrose and sodium chloride, and newborn acidifying woods Ge Shi intravenous vehicles comprises liquid and nutritional supplement, electrolyte supplements, as based on the fill-in of woods Ge Shi dextrose etc.Also can there be antiseptic and other additive, as antimicrobial, antioxidant, chelating agen, somatomedin and noble gas etc.
Usually, term used herein " treatment " etc. refers to influence object, tissue or cell to obtain required pharmacology and/or physiological action.This effect can be preventative, i.e. prevent disease or its symptom or symptom wholly or in part, and/or can be curative, i.e. cure diseases partially or completely.Any treatment or prevention that term used herein " treatment " has covered the disease of vertebrates, mammal, particularly people, comprise that prevention may be tended to disease takes place but yet diagnosis suffer from this sick object this disease take place; Suppress this disease, promptly block its development; Perhaps alleviate or improve the effect of this disease, cause that promptly disease effector goes down.
The present invention includes the application of the various pharmaceutical compositions that are used to improve disease.By with BTC-δ 4, the form that is fit to give with carrier, excipient and additive or adjuvant object is merged in the combination of its analog, derivant or salt and one or more pharmaceutically active substances or BTC-δ 4 and one or more pharmaceutically active substances, prepares the pharmaceutical composition of one embodiment of the present invention.
Carrier or adjuvant commonly used comprise magnesium carbonate, titanium dioxide, lactose, mannitol and other sugar, Pulvis Talci, milk protein, gelatin, starch, vitamin, cellulose and its derivant, animal and plant oil, Polyethylene Glycol and solvent, as sterilized water, alcohol, glycerol and polyhydric alcohol.Intravenous vehicles comprises liquid and nutritional supplement.Antiseptic comprises antimicrobial, antioxidant, chelating agen and noble gas.Other pharmaceutically acceptable carrier comprises that aqueous solution, non-toxic excipients comprise salt, antiseptic, buffer etc., as " " Lei Mingdun pharmaceutical science " (Remington ' sPharmaceutical Sciences), the 20th edition, Williams and Wilkins (2000) and " british national formulary " (TheBritish National Formulary) the 43rd edition, (British Medical Association and RoyalPharmaceutical Society of Great Britain, 2002; Http:// bnf.rhn.net) described, this paper is for referencial use with fitting in it.Adjust the pH and the accurate concentration of the various components of pharmaceutical composition according to this area routine techniques." pharmacological basis of treatment " (The Pharmacological Basis forTherapeutics) (the 7th edition, 1985) referring to Goodman and Gilman.
Preferably with the preparation of the form of dosage unit with give pharmaceutical composition.Solid dosage unit comprises tablet, capsule and suppository.In object treatment, age and body weight according to the characteristic of compound activity, administering mode, disease and the order of severity, object can adopt different daily doses.Yet in some cases, higher or lower daily dose may be suitable.Can give the form of single dosage unit or several smaller dose units by single, also can give daily dose by the dosage that repeatedly separates with specific interval.
Can the part or whole body treat the pharmaceutical composition of the present invention of effective dose.Certainly, the effective dose that reaches this purpose depends on the order of severity of disease and the body weight and the overall state of object.Usually, the consumption that external used dosage can be the administration of pharmaceutical composition original position provides with instructing, and available animal model is determined the effective dose of treatment cytotoxicity side effect.For example, Langer, Science, 249:1527 has described various considerations in (1990).Peroral dosage form can be the form of hard gelatin capsule, and wherein active component and inert solid diluent are mixed as calcium carbonate, calcium phosphate or Kaolin.They also can be the forms of Perle, and wherein active component and water or oil medium are as Oleum Arachidis hypogaeae semen, liquid paraffin or mixed with olive oil.
Aqueous suspension contains active substance usually, is mixed with the excipient that is fit to produce aqueous suspension.This excipient can be a suspending agent, as sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl emthylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and Radix Acaciae senegalis; Dispersant or wetting agent can be the phospholipid such as the lecithin of (a) natural generation; (b) condensation product of alkylene oxide and fatty acid, for example, Myrj 45; (c) condensation product of oxirane and long-chain fatty alcohol, for example, 17 carbon ethyleneoxy hexadecanols; (d) oxirane and condensation product derived from the partial ester of fatty acid and hexitol, as the polyoxyethylene sorbitol monoleate, or (e) oxirane and condensation product derived from the partial ester of fatty acid and hexitan, as Tween-81.
Pharmaceutical composition can be the form of sterile injectable aqueous or oiliness suspension.Can prepare this suspension with suitable dispersant or wetting agent and aforesaid suspending agent according to known method.Aseptic injection also can be sterile injectable solution or the suspension in outer acceptable diluent of nontoxic gastrointestinal tract or the solvent, as the solution of 1,3 butylene glycol.Adoptable acceptable carrier and solvent are water, Ringer's mixture and isotonic sodium chlorrde solution.In addition, aseptic fixed oil is usually as solvent or suspension media.Therefore, any nonirritant fixed oil be can adopt, synthetic monoglyceride or diglyceride comprised.In addition, fatty acid such as oleic acid can be used for preparing injection.
Also can be in the form of liposome delivery system, as giving The compounds of this invention in little unilamellar vesicle, big unilamellar vesicle and the multilamellar vesicle.Can form liposome as cholesterol, octadecane amine stearylamine or phosphatidylcholine by various phospholipid.
Usually about the order of magnitude of every kg body weight 0.5mg-20mg, the preferred dosage scope is every kg body weight about 0.5mg-10mg every day (every patient's every day of about 0.5g-3g) to the dosage level of BTC-δ 4.Can will change because of treated host and concrete administering mode with the absorption of active ingredient that produces single dose with the carrier combination of materials.For example, the preparation that is used for the human oral administration can contain the carrier material of have an appointment 5mg-1g reactive compound and suitable and convenient amount, and the carrier material can account for the 5-95% of total composition.Dosage unit form contains the active component of the 5mg-500mg that has an appointment usually.
Yet should understand, the given dose level that is used for concrete patient depends on various factors, comprises activity, age, body weight, health status, sex, diet, administration time, route of administration, excretion rate, the drug regimen of used specific compound and the order of severity of the disease specific for the treatment of.
In addition, polypeptide of the present invention can make up with other chemical compound, so that the operability combination to be provided.Be intended to comprise any chemical compatibility combination of pharmaceutically active substances, as long as the activity of BTC-δ 4 is not eliminated in this combination.
Measure the external model of BTC-δ 4 effects
The differentiation of AR42J-B20 cell
The cell-derived pancreas tumor from chemical induction of pancreas AR42J is expressed external secretion and neuroendocrine characteristic (Rosewicz etc., 1992).After activin A processing, the AR42J-B20 cell stops growing, and its form significantly changes (neurite extension).In addition, the cellular expression of activin-processing coding GLUT2, ATP-sensitivity K +The mRNA of passage and pancreatic polypeptide (PP).Therefore, activin A changes the AR42J cell into endocrine cell.
Handle these cells with BTC-δ 4 and activin A and they further can be converted into the cell that produces insulin.The insulin that can behind the anti-insulin antibody staining cell, produce in response to BTC-δ 4 with determination of immunofluorescence method.Perhaps, available AR42J clone AR1898-0192 quantitative assay AR42J cell is to the conversion of insulin secretory cell.This clone contains secreting type alkali phosphatase (SEAP) gene, and it is positioned at rat insulin II gene promoter downstream.Cause the synthetic of alkali phosphatase and be secreted into culture medium with BTC-δ 4 these cells of stimulation, available Enzymology method is measured.
The epithelial cell line of immortalization
Pancreas precursor system separates from the pancreas conduit (Sharma etc., 2001) that carries the antigenic transgenic mice of responsive to temperature type SV40 T (" immortalization mice (immortomice) ").When cultivating the cell of these mices for 33 ℃, express SV40 T antigen, cell becomes the conditional immortalisation cell; Yet at 37 ℃, the T antigen presentation is closed, and cell suitably breaks up.Therefore, these cells do not demonstrate island/neuroendocrine granule when cultivating for 33 ℃, can not express the island labelling.Yet, 37 ℃ cultivate about 2 weeks after, during about 50% cell death, all the other cells begin to be divided into and demonstrate primary island sample insulin and the particulate cell of glucagon.Be transplanted to the following time of the scrotum of nude mouse, cultured cells all develops and conduit sample and island sample phenotype under two kinds of temperature.
This cell line is used for the test (similar to above-mentioned test) of rapid sensitive, to measure the differentiation of insulin expression and beta cell.In this case, with rat insulin 1 promoters driven green fluorescent protein (GFP) receptor expression.This cell line provides very sensitive method, identifies beta cell differentiation factor such as BTC-δ 4 by the GFP fluorescence of measuring in the living cells, and described beta cell differentiation factor stimulates the expression of beta cell specific gene such as insulin.
The body inner model of type 2 diabetes mellitus
Except that streptozotocin (STZ) model (referring to embodiment 7), many other animal models of type 2 diabetes mellitus all can be used for the activity in vivo of test b TC-δ 4.These models comprise that operation is as the type 2 diabetes mellitus model (Bonner-Weir etc., 1983) of the pancreatectomy generation of rat and the genetic model of feeding based on selectivity.Genetic model comprises Goto-Kakizaki (GK) rat, spontaneous diabetes Torii (SDT) rat, Otsuka-Long-Evans-Tokushima obesity (OLETF) rat, db/db mice and NOD/Ltj mice.
Goto-Kakizaki (GK) rat demonstrates metabolism, hormone and the angiopathy similar to people's diabetes finding.Unlike many other Rodents models of type 2 diabetes mellitus, the GK rat is not fat.The feature of Goto-Kakizaki rat comprises the fasting hyperglycemia, in vivo with isolating pancreatic cell in impaired in response to the insulin secretion of glucose, and the insulin resistance of liver and periphery.Late complication such as retinopathy, microangiopathy, neuropathy and nephropathy have been described in the document.
In spontaneous diabetes Torii (SDT) rat model, glucose spontaneously takes place and does not tolerate in male rat after 14 ages in week, and insulin secretion is impaired simultaneously; Take place with significantly hyperglycemia and the significantly diabetes of hypoinsulinemia after 20 ages in week.The feature of diabetes Long-Evans rat is after 18 ages in week the hyperglycemia that moderate is fat and fall ill late period to take place, and the complication relevant with chronic diabetes.Though identified a plurality of locus by genetic analysis, caused the reason insulin resistance and the impaired combination of insulin secretion seemingly of diabetes in these rats, the anthropoid type 2 diabetes mellitus of class.Otsuka-Long-Evans-Tokushima obesity (OLETF) rat-derived is from insulin resistance and the impaired spontaneous combination of insulin secretion, the anthropoid type 2 diabetes mellitus of class.In these rats, insulin sensitivity reduces with old and feeble, promptly compares with contrast Long-Evans Tokushima Otsuka (LETO) rat that mates age in week, and is normal when 6 ages in week, and reduces by 80% reduced for 40%, 18 age in week when 12 ages in week after.Insulin secretion is in 40 weeks during ages impaired (14), may participate in the island dysfunction morbidity of the too high OLETF rat of triglyceride to the fat toxicity (lipotoxicity) of island beta cell.In addition, compare with the LETO rat, the regeneration capacity of the pancreas beta cell of OLETF rat reduces (17).Because the chronic progressive external form of diabetes, OLETF rat are applicable to the pathophysiology during the research prediabetes and change.
Carry point mutation in thin hormone (leptin) acceptor gene of db/db mice, birth back 8-10 week, the spontaneous blood sugar level that takes place raise and the depletion of pancreas β cell.About 4 weeks during ages these mices also become significantly fat.
The feature of NOD/Ltj mice is: insulitis, the leukocyte infiltration of islets of langerhans.Be about the female of 12 ages in week, the insulin content of pancreas significantly reduces, and male this phenomenon takes place after several weeks.The sign of onset diabetes is that the plasma glucose of moderate glycosuria and not fasting is higher than 250mg/dl.Hypoinsulinism of diabetes NOD/Ltj mice and hyperglycemia, this shows that beta Cell of islet is destroyed by selectivity.The NOD/LtJ mice is a polygenes to the susceptibility of IDDM, and environment comprises that raising condition, health status and diet have intense influence to penetrance.NOD/LtJ is female more extensive than male application, because the generation of IDDM symptom more early and sickness rate higher (90-100% during 30 ages in week).The frequency of the 30-40 male generation of NOD/LtJ IDDM during age in week is 40-60%.Male mice can be used for some to be used, and comprises " quickening to shift " and some in vitro studies of drug research, IDDM.The key component of the diabetes susceptibility of NOD mice is unique MHC haplotype (H2 G7=K d, Aa d, Ab G7, E Null, D b).The NOD mice also represents multiple unusual immunophenotype, it is impaired that the adjusting defective, NK cell function defective, macrophage that comprises antigen presenting cell immunoloregulation function defective, T B lymphocyte repertoire produces the defective (Fan etc., 2004) and the wound healing of cytokine.They also lack α hemolytic complement component C5.The audition of NOD/LtJ mice also is badly damaged.The transgenic that will cause various sudden changes, the cytokine gene targeting sudden change of immunodeficiency and influence immunologic function is backcrossed in the NOD/Lt inbreeding strain background.
The obtainable test the present invention of those skilled in the art infers polypeptide and comprises to be used for other appropriate method of the present invention: adopt above-mentioned type 2 diabetes mellitus body inner model, test beta cell regeneration, improve glucose and do not tolerate and improve the control of blood glucose (fasting serum glucose), when blood sugar level raises, stimulate insulin secretion and the glucagon suppression secretion, hypoglycemia and improvement such as intravenous glucose tolerance test, arginine stimulation and the hyperglycemia of measuring beta cell function sign clamp.These tests also can be applicable to the human clinical trial.
Below will be by describing the present invention in detail with reference to the mode of following non-limiting example and accompanying drawing.
Embodiment 1: Detect BTC-δ 4 and be cloned into pBluescript II SK with RT-PCR
For a short time (Vic. Australia) separates MCF-7 cell that 70-80% converges and total RNA of human breast skin flbroblast for Qiagen, Clifton Hill according to manufacturers instruction to take out test kit with Rneasy.Prepare normal human breast skin flbroblast by a skin that obtains in the breast reduced operation.With the DMEM that is supplemented with 10% hyclone and penicillin-streptomycin sulfate this skin was cultivated 5 days as explant, grown up to monolayer up to fibroblast.Then, according to manufacturers instruction with Superscript II enzyme, (Gibco BRL, Gaithersburg are cDNA with total RNA (2 μ g) reverse transcription MD) to oligomerization dT primer.Increase corresponding to the cDNA of people BTC-β with sense primer and antisense primer with PCR.
Sense primer be (5 ' CTCGTCGACGAGCGGGGTTGATGGACCGG-3 ') (SEQ ID NO:3)
Antisense primer be (5 ' CTCCTGCAGTTAAGCAATATTTGTCTCTTC-3 ') (SEQ ID NO:4).
The nucleotide correspondence of underscoring is in SalI (sense primer) and PstI (antisense primer) restriction site.With 50 μ l60mM Tris-SO 4, 18mM (NH 4) 2SO 4, 1.5mM MgSO 4(pH9.1), (Gibco BRL, Gaithersburg MD) carry out PCR with 1 μ l cDNA for each primer of 0.2mM dNTP, 200ng, 1U eLONGase.At the beginning 94 ℃ hatch 3 minutes after, carry out 35 following circulations: 94 ℃ 1 minute, 50 ℃ 1 minute, 68 ℃ 1 minute, extended 4 minutes at 68 ℃ at last.Separate 416bp BTC-δ 4 PCR products with 2% agarose gel, with under uviol lamp, observing behind the ethidium bromide staining.(Promega, Madison WI) as the molecular size standard, the results are shown in Figure 1A to the PCR labelling.Behind the electrophoresis, by 0.4M NaOH with gel Southern transfer to positively charged nylon membrane (Roche, Castle Hill, NSW, Australia) on.At prehybridization buffer (5 * SSC, 5 * Denhardt, 0.1% sodium lauryl sulphate (SDS) and 50 μ g/ml salmon sperm DNAs) in 55 ℃ hatched this trace 2 hours, containing useful Gigaprime test kit (Geneworks, Adelaide Australia) produces with random priming 32The people BTC cDNA probe (D of P-dCTP-labelling 32-Y 111) same buffer in hybridization 16 hours.With 2 * SSC, washing is 5 minutes twice under the 0.1%SDS room temperature, uses 0.5 * SSC then, and 0.1%SDS washs 15 minutes twice at 55 ℃ again.Then-80 ℃ with this trace exposure Kodak XAR X-line film, the results are shown in Figure 1B.
Clone by the following method and check order BTC and BTC-δ 4 PCR products: by the agarose gel electrophoresis separated product, with the Concert test kit (Gibco BRL, Gaithersburg, MD) carry out the gel extracting after, reclaim fragment.Then with the PCR product of SalI and PstI digestion purification, and sub-clone is gone into the pBluescript IISK of SalI/PstI-digestion, and (Strategene, La Jolla CA), produce pBlue-BTC and pBlue-BTC-δ 4, and introduce e. coli jm109.The amplification recombiant plasmid is also used the terminal method order-checking of dideoxy nucleotide chain, and the Thermosequenase cycle sequencing test kit (Amersham-Pharmacia Biotech, Sydney, AUS) that contains M13 T7 and T3 promoter primer is adopted in order-checking.Order-checking PCR product on both direction is with the checking sequence; Two independent clonings are carried out twice order-checking, the results are shown in Figure 2A and B.
Embodiment 2: With escherichia coli expression and purification of Recombinant BTC and BTC-δ 4
In order to characterize the biologic activity of BTC-δ 4, we are with corresponding cDNA (Asp 32-Ala 129No signal peptide) being cloned into the pET-32a expression vector, is the thioredoxin fusions with this protein expression in e. coli bl21 trxB (DE3).The thioredoxin reductase defective of this bacterial strain can form disulfide bond in kytoplasm.The sketch map of used construction is seen Fig. 5 A.BTC (Asp in bacterial expression vector pET3.2a 32-Tyr 111) or total length BTC-δ 4 (Asp 32-Ala 129, deduct hydrophobic signal peptide Met 1-Ala 31) mature form be expressed as the thioredoxin fusion rotein.PET3.2a-BTC and pET3.2a-BTC-δ 4 constructions are transformed into coli strain BL21 (DE3), and IPTG induces the back expressing protein.After inducing, collecting cell and cracking are with Ni-NTA agarose and RP-HPLC purification BTC or BTC-δ 4.After the expression, cell lysis is with Ni-NTA agarose purified fusion protein.With the thioredoxin fusion rotein of enterokinase cutting purification,, use reversed-phase HPLC purification BTC-δ 4 to homogeneous to discharge BTC-δ 4.
By PCR with pBlue-BTC-δ 4 (Dunbar etc., 2000) for template with following primer sets amplification BTC-δ 4 32-129Open reading frame sequence: 5 '-CGT CCATGGCTGATGGGAATTCCACCAGAAGT-3 ' (SEQ ID NO:5) (justice) and 5 '-CGT CTCGAGTCATTAAGCAATATTTGTCTCTTC-3 ' (SEQ ID NO:6) (antisense).NcoI and XhoI recognition site (underscore) are connected to justice and antisense primer.Also use the aminoacid 32-111 (BTC of pET system expression and purification BTC 32-111), as positive control, (Seno etc., 1996) as mentioned above, or be produced as the thioredoxin fusion rotein with plasmid pET32a.In this construction, by PCR with pBlue-BTC-δ 4 (Dunbar and Goddard, 2000) for template with following primer sets amplification BTC 32-111The ORF sequence: 5 '-CGT CCATGGCTGATGGGAATTCCACCAGAAGT-3 ' (SEQID NO:7) (justice) and 5 ' CGT CTCGAGTCAGTAAAACAAGTCAACTGT-3 ' (SEQ ID NO:8) (antisense).Also NcoI and XhoI recognition site (underscore) are connected to justice and antisense primer.After the amplification, with NcoI/XhoI digestion PCR product, and be cloned into the pET32a carrier that NcoI/XhoI digests, as mentioned above.Keep the plasmid pETBTC-δ 4 that obtains with e. coli jm109 32-129And pETBTC 32-111, be transformed into e. coli bl21 trxB (DE3) cell and express.In order in BL21trxB (DE3) cell, to express BTC or BTC-δ 4, single colony inoculation in the 50ml culture, with 37 ℃ of overnight incubation of LB culture medium (being supplemented with 50 μ g/ml ampicillin and 15 μ g/ml kanamycin), is constantly vibrated.Then, with the fresh LB culture medium of 10ml this overnight culture inoculation 200ml.At 37 ℃ of cultured cells to OD 600nmBe 0.4-0.5, use IPTG (1mmol/l) to induce then, centrifugation after 3 hours (4000rpm, 4 ℃, 10 minutes) is stored in-80 ℃ before the purification.
For purification is expressed as the BTC or the BTC-δ 4 of fusion rotein, at the cell precipitation of melting chilling on ice, be resuspended in the 18ml BugBuster protein extraction agent that contains lysozyme (100 μ g/ml), gentle vibration was hatched 10 minutes under the room temperature.After hatching, supersound process (3 * 5 pulse per second (PPS)) cell lysate to be reducing viscosity, centrifugally makes its clarification (20 minutes, 16000rpm, 10 minutes).After centrifugal, clarifying cell lysate is adjusted in the 10mmol/l imidazoles, hatched with 6ml Ni-NTA agarose, at 50mmol/lNaH 2PO 4, 4 ℃ of pre-equilibrations 1 hour among the 0.3mol/l NaCl, 10mmol/l imidazoles (pH8.0), gentle vibration.After hatching, centrifugal resin (5 minutes, 15000rpm, 4 ℃) is removed supernatant, by being suspended in 50mmol/l NaH 2PO 4, 0.3mol/l NaCl, 40mmol/l imidazoles (pH8.0) washing resin.After carrying out three above-mentioned washings continuously, by at 12ml 50mmol/l NaH 2PO 4, hatch 15 minutes elute proteins of resin among the 0.3mol/l NaCl, 250mmol/l imidazoles (pH8.0).Contain BTC or BTC-δ 4 in the supernatant that obtains, change the buffer of enterokinase cutting several times (50mmol/l Tris-Cl, 1mmol/lCaCl then 2, 0.1% tween 20, pH7.4) dialyse this supernatant spend the night (Spectra/Por, 3.5kDa MWCO, SpectrumLaboratories).In order to remove the thioredoxin fusion partner of BTC or BTC-δ 4, add enterokinase, making final concentration is 0.1 unit/20 μ g protein, and 37 ℃ of gentle ground overnight incubation of mixing.Further separate BTC or BTC-δ 4 and thioredoxin fusion partner by above-mentioned Ni-NTA agarose affinity chromatography.In this case, catch the thioredoxin fusion partner of cutting on resin, BTC or BTC-δ 4 are enriched in and flow through in the component.Be further purified with reversed-phase HPLC and flow through BTC or the BTC-δ 4 that exists in the component.Briefly say, use 0.1%TFA1: 4 dilute the Ni-NTA agarose (v/v) flows through component, and it is put on C4 Prep-Pak reversed-phase HPLC post (25mm * 100 mm; 300 , 15 μ m; Millipore-Waters), flow velocity is 10ml/ minute.Wash this post with 0.1%TFA, up to OD 214nmGet back to baseline, this post of acetonitrile eluting of using 150 minutes then in the presence of 0.08%TFA with 8-80% gradient (v/v), flow velocity is 10ml/ minute.Collect the 20ml component, with SDS-PAGE and the anti-people BTC of goat extracellular domain (Asp 32-Tyr 111) each five equilibrium of Western engram analysis (50 μ l) of antibody, contain the component of pure BTC or BTC-δ 4 with evaluation.Set contains the component of BTC or BTC-δ 4, with analyzing RP-HPLC, electronic spraying ionization massspectrum and SDS-PAGE purity assay.(2.1 * 100mm) (Alltech) analyzed RP-HPLC with the acetonitrile and the 0.08%TFA of 8-80% linear gradient (v/v), and flow velocity is 0.5ml/ minute by Brownlee aquapore C4RP-HPLC post with 40 minutes.The results are shown in Figure 5B.
Reorganization BTC that the electronic spraying ionization massspectrum is measured and the molecular mass of BTC-δ 4 are respectively 9211.35 ± 0.29Da and 11450.26 ± 0.23Da, and be consistent with 9249 and the theory of computation quality (data not shown) of 11452Da.After SDS-PAGE and silver dye, obtain to be about the single band of 9kDa and 11.5kDa, they are corresponding to detected BTC and BTC-δ 4 under reduction or the non-reduced condition.Further verified the purity of BTC and BTC-δ 4 by N-terminal sequence analysis (5 circulations), this analysis provides the N-end sequence of expectation, purity approximately＞95%.
Perhaps, with BTC-δ 4 32-129(containing initial methionine residues) is cloned into expression vector pET3b, and the recombiant protein dissolving is folding again from occlusion body, with cation exchange column and solvent resistant column chromatogram purification, (Maeda etc., 2002) as previously mentioned.In this research preparation all recombiant proteins all by lyophilizing and be stored in-80 ℃ stand-by.
Embodiment 3: Structure is used for BTC-δ 4 expression plasmids that mammal is expressed
Total length BTC-δ 4 (1-129) is cloned into carrier pcDNA3.1 (Invitrogen), produces the expression vector that is used for mammal generation BTC-β (as described below).Briefly say, with the pBlue-BTC-δ 4 of ApaI and BamHI digestion embodiment 1, the insert that purification discharges behind the agarose gel electrophoresis.To digest then and the insert of purification is cloned among the pcDNA3.1 of ApaI/BamHI-digestion, and be transformed in the escherichia coli TOP10 cell (Invitrogen) back and identify recombinant clone.(wherein Flag epi-position DYKDDDDK inserts aminoacid S in order to produce " the Flag-labelling " pcDNA3.1-BTC-δ 4 constructions 35-T 36Between, as shown in Figure 3), pBlue-BTC-δ 4 as pcr template, is adopted following primer:
5 '-CTCGG GAATTCC
Figure A20058003241900301
TCCTGAA-3 ' (SEQ ID NO:9) (justice; The nucleotide correspondence of underscore is in the EcoRI restriction site; The nucleotide coding FLAG epi-position label of double underline) and
5 '- CTCCTGCAGTTAAGCAATATTTGTCTCTTC-3 ' (SEO ID NO:10) (antisense; The nucleotide correspondence of underscore is in the PstI restriction site).The PCR product that purification obtains is with EcoRI and PstI digestion and be cloned among the pBlue-BTC-δ 4 of EcoRI/Pst1-digestion.Then, digest pBlue-BTC-δ 4 constructions of the Flag-labelling that obtains, and be cloned into the pcDNA3.1 of ApaI/BamH1-digestion, produce pcDNA3.1-FLAG-BTC-δ 4 with ApaI/BamH1.
Embodiment 4 In the COS-7 cell, express and analyze the construction of BTC and BTC-δ 4 FLAG-labellings
As other member of EGF family, BTC synthesizes the precursor protein (pro-BTC) of striding film-grappling, and it can produce after the proteolysis cutting and contain EGF-motif (Asp 32-Tyr 111) solubility BTC.Kept the hydrophobicity signal peptide and do not had the membrane spaning domain explanation, BTC-δ 4 may be a secretory protein.In order to check this inference, the complete open reading frame of engineered BTC or BTC-δ 4 is with at aminoacid Ser 35And Thr 36Between additionally mixed the FLAG epi-position, be used for 3 described subsequent detection as embodiment, this open reading frame is cloned into expression vector pcDNA3.1, monitoring secretion behind the transient transfection COS-7 cell.After the transfection 72 hours, collect cleer and peaceful cell lysate on the culture, and existence excretory with the Western engram analysis with ELISA and BTC-FLAG that links to each other with cell and BTC-δ 4-FLAG.
In the transient transfection of pcDNA3.1-FLAG-BTC-δ 4 and pcDNA3.1-FLAG-BTC, with DMEM/10%FBS with the COS-7 cell inoculation in 12 orifice plates, density is 2 * 10 5Cells/well.After the overnight incubation, with Lipofectamine 2000 and Optimem-1 culture medium (all from Invitrogen) according to manufacturer's description with 2 μ g construction DNA transfectional cells.After 12 hours, washed cell twice replenishes fresh Optimem-1 culture medium.After the transfection 72 hours, collect culture medium (CM), the preparation cell lysate.Make CM clarification by centrifugal, with the existence of BTC or BTC-δ 4 in the ELISA of anti-FLAG-M2 antibody (Sigma) or the Western engram analysis culture medium.The BTC or the BTC-δ 4 that exist in the Western engram analysis cell lysate with anti-FLAG-M2 antibody.Remove behind the CM with PBS washed cell twice, then with the direct cell lysis of the sample buffer of SDS-PAGE and be heated to 95 ℃ 5 minutes, thereby prepare cell lysate.
In the elisa assay of CM, 90 μ l CM and 10 μ l 10 * bag are cushioned liquid (0.15mol/l Na 2CO 3, 0.35mol/l NaHCO 3, pH9.3) mix, add in the 96 hole immunoadsorption plates.Plate is spent the night at 4 ℃ of bags, uses the 2%BSA sealing in the PBS/0.1% tween (PBS-T) then.Wash plate 4 times with PBS-T, hatched 1 hour at 37 ℃ with the anti--FLAG antibody of PBS-T (2.5 μ g/ml) dilution then.After hatching, wash plate as mentioned above, hatch (the link coupled sheep anti-mouse antibodies of HRP-is from Silenus Laboratories) with the link coupled sheep anti-mouse antibodies of HRP-(1: 2000) of PBS-T dilution.Wash plate 4 times with PBS-T then,, use 2mol/l H with o-phenylenediamine (OPD) substrate color development 2SO 4Stop.Read absorbance at 490nm.The result is expressed as the mean+SD (SD) of three replications.For existence with BTC or BTC-δ 4 on the elisa assay cell surface, remove CM, use PBS washed cell 3 times.Use 4% paraformaldehyde fixed cell then, with anti--FLAG M2 antibody staining, as mentioned above.
In with anti--Western engram analysis that FLAG M2 antibody carries out, differentiate CM or cell lysate with SDS-PAGE (10-20%Tris-Tricine gel).Then protein transduction is moved on to celluloid (Hybond-Cextra; Amersham) on, with mouse anti-FLAG M2 antibody (2.5 μ g/ml), use the link coupled sheep anti-mouse antibodies of HRP-(1: 10000) to detect film subsequently.Observe the protein of HRP-labelling with Supersignal West Dura Extended Duration Substrate (Pierce).
Brief summary finishes fruit among Fig. 6.Shown in Fig. 6 A and B, with the Western trace (～14kDa) or enzyme immunoassay (EIA) (EIA) clearly detect BTC-δ 4-FLAG in the culture supernatant, in the cell lysate or considerably less (Fig. 6 A and the B) that exist on the cell surface.On the contrary, BTC-FLAG mainly be present in the cell lysate (～20kDa) and on the cell surface (Fig. 6 A and B).Therefore, be that cell surface extracellular domain cutting back is excretory unlike the pro-BTC of film-grappling, BTC-δ 4 lacks membrane spaning domain, seemingly expresses the back direct secretion.
The ErbB-receptors bind of embodiment 5:BTC-δ 4 and short mitotic activity
In order to study the biologic activity of BTC-δ 4, originally we tested it stimulates the ability of the Balb/c3T3 l cell propagation of expressing ErbB1.(Dunbar etc., 1999) measure BTC and the fibroblastic short mitotic activity of 4 couples of Balb/c 3T3 of BTC-δ as previously mentioned.Balb/c 3T3 cell is available from American type culture collection.
The results are shown in Figure 7A and 7B.As expected, BTC stimulates Balb/c 3T3 propagation in dosage dependence mode.On the contrary, BTC-δ 4 does not stimulate cellular proliferation, even concentration is up to 100nmol/l.In addition, the not effect of antagonism BTC of BTC-δ 4 in this cell line.
By measure on BTC or BTC-δ 4 competitive ErbB1 of replacement or the ErbB4 receptor [ 125I]-ability of the recombinant human B TC of labelling determines the binding affinity of BTC and BTC-δ 4 and ErbB receptor, described ErbB1 or ErbB4 receptor are present in Chinese hamster ovary (CHO) cell (the CHO-ErbB4 cell of AG2804 fibroblast (Dunbar etc., 1999) or ErbB4 stable transfection respectively; (Tzahar etc., 1996), the YosefYarden professor friendship present of Israel Ci Man Wei institute) on.In order to finish this mensuration, in the 24-orifice plate, AG2804 or CHO-ErbB4 cell culture are converged to 70-80% with DMEM/10%FBS.(people's lung fibroblast.Use then binding buffer liquid (100mmol/l Hepes, (pH7.6), 120mmol/l NaCl, 5mmol/l KCl, 1.2mmol/l MgSO 4, the 8mmol/l glucose, 0.1%BSA) washed cell twice, then with [ 125I]-(10000-15000cpm uses chloramine-T with Na[to rhBTC 125I] labelling, be about 20 μ Ci/ μ g to specific activity) and the unmarked BTC that raises of concentration or BTC-δ 4 (the AG2804 cell is 0-100nmol/l, and the CHO-ErbB4 cell is 0-10nmol/l) together in binding buffer liquid 4 ℃ hatched 18 hours.Use the cell three times of ice-cold Hank ' s buffer salt solution washing labelling then, and with 1ml 0.5mol/l NaOH/0.1% (v/v) Triton X-100 cracking 30 minutes.Use γ-enumerator (Wallac1470) to measure the radioactivity of cell lysate subsequently.Under the excessive 100 times situation of unmarked part, carry out this biologic test and measure non-specific binding.Non-specific binding generally is about 5% of total binding.
We have also compared the ability of the tyrosine phosphorylation of BTC and BTC-δ 4 stimulation ErbB1 or ErbB4.In the 10cm culture dish with the cell line AG2804 of overexpression ErbB1 receptor or Chinese hamster ovary celI (the CHO-ErbB4 cell of overexpression ErbB4 receptor; The present of the Yosef Yarden of Israel Ci Man Wei institute professor friendship) is cultured to and converges, hatched 12-14 hour with serum-free medium then, use 10nmol/l BTC, BTC-δ 4 or the combination of the two irritation cell 10 minutes at room temperature then.After the stimulation, with twice of PBS washed cell, use lysis buffer (0.5ml) (50mmol/l Tris-Cl pH7.4 subsequently, 150mmol/l NaCl, 1% deoxycholic acid, 1% TritonX-100,0.1%SDS, the 5mmol/l sodium orthovanadate, 10mmol/l sodium fluoride, 1mmol/l EGTA and adequate proteins enzyme inhibitor TM) cracking.Make cell lysate clarification by centrifugal (4 ℃ of 15000g are centrifugal 20 minutes), respectively by with lysate with 1 μ g rabbit polyclonal anti--ErbB1 antibody (1005) (Santa Cruz) or rabbit polyclonal be anti--ErbB4 antibody vibrates 4 ℃ of gentlenesses and hatch 2 hours immunoprecipitation ErbB1 or ErbB4.
After hatching, add albumen-G agarose, hatched this mixture 1 hour at 4 ℃ again.Centrifugal collection immune complex is with lysis buffer washing three times and heating (3 minutes, 95 ℃) in the SDS-PAGE sample buffer.With SDS-PAGE or 10-20%Tricine gel separation protein, transfer to nitrocellulose filter (Hybond C, Amersham) on.At first, survey with the link coupled sheep anti of HRP--mouse antibodies then with anti--phosphorylated tyrosine monoclonal antibody (PY20) detection membrane.(Amersham) observe the HRP-labelled protein with enzyme-Lian chemiluminescence (ECL).In order to verify that applied sample amount equates, the elution trace, with rabbit polyclonal anti--ErbB1 or rabbit polyclonal be anti--ErbB4 antibody and the link coupled rabbit of HRP-be anti--sheep antibody surveys again.Anti--ErbB1 (1005), anti--ErbB4 (C-18) and anti--phosphorylated tyrosine (PY20) antibody is available from Santa Cruz, and the link coupled rabbit of HRP-resists-and sheep antibody is available from Zymed Laboratories.
Brief summary finishes fruit among Fig. 7 C-7F and Fig. 8.We find, BTC but not BTC-δ 4 can replace people's lung fibroblast (AG2804) go up the ErbB1 receptor that exists [ 125I]-BTC (Fig. 7 C and D), and induce ErbB1 receptor autophosphorylation phosphorylation (Fig. 8).Similarly, BTC but not BTC-δ 4 competitions are in conjunction with ErbB4 receptor (Fig. 7 E and F) and induce ErbB4 receptor tyrosine phosphorylation (Fig. 8).These results show that opposite with BTC, BTC-δ 4 does not demonstrate the affinity with ErbB1 or ErbB4.And BTC-δ 4 does not activate ErbB1 or ErbB4.
These results are all beyond one's expectations.Do not stimulate cellular proliferation because this means BTC-δ 4, our discovery prompting, BTC-δ 4 induces risk low than the cancer of real BTC.As and if BTC-δ 4 brings into play its effect by a kind of receptor of identifying not yet, rather than by ErbB1 or ErbB4 receptor.Available methods described herein, or proceed further research with BIAcore test.
Embodiment 6: Measure the differentiation and the apoptosis of AR42J cell
Known natural B TC can stimulate the differentiation of pancreas beta cell (Mashima etc., 1996; Watada etc., 1996; Yamamoto etc., 2000; Li etc., 2001; Li L etc., 2003; Li etc., 2004), some evidence promptings are arranged, (Ishiyama etc., 1998) may take place by unique non-ErbB cell surface receptor in this effect.
For the influence (as by estimating inducing insulin expression) that detects 4 pairs of pancreas beta cell differentiation of BTC-δ, we have adopted pattern cell line AR42J-B20, and it is the sub-clone of AR42J, is a kind of pancreas tumor cell line of secreting amylase.In these cells, BTC and activin A synergism change them into insulin secretory cell (Mashima etc., 1996).Therefore, activin A changes the AR42J-B20 of secreting amylase into produce pancreatic polypeptide (PP) endocrine cell.Activin A also induces these apoptosis, and when not having survival factors, the cell of many activins-processing is the death (Furukawa etc., 1999) by apoptosis after changing the cell that produces PP into.
BTC brings into play two kinds of effects in the AR42J-B20 cell: at first, it suppresses the inductive apoptosis of activin A, and secondly, it changes them into produce insulin cell (Mashima etc., 1996; Furukawa etc., 1999).With the DMEM culture medium culturing AR42J-B20 cell that contains 10% hyclone, (Mashima etc., 1996) as previously mentioned.
In order to estimate differentiation, cell was hatched 48 hours with 2nmol activin A and 1nmol BTC or BTC-δ 4 to the cell that produces insulin.Fixed cell is used the anti-insulin-antibody staining then, (Mashima etc., 1996) as previously mentioned, counting insulin-positive cell.Dye nuclear with DAPI.
(Wako Pure Chemicals, Osaka Japan) estimate apoptosis with terminal deoxynucleotidyl transferase (TUNEL) technology.Use 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyl bromination tetrazolium (MTT) is estimated the change (Carmichael etc., 1987) of living cells quantity.
Shown in Fig. 9 A, during colored printing, the AR42J-B20 cellular response is divided into the cell that produces insulin in the combination of activin A and BTC.Quantitatively, (meansigma methods ± S.E. n=4) becomes the insulin positive to 78 ± 6.5% cell after 48 hours.The cell that activin A and BTC handle has the process of prolongation and expresses immunoreactive insulin, shown in Fig. 9 C during as colored printing.Cell with the combined treatment of activin A and BTC-δ 4 also becomes the insulin positive; The cell of 72 ± 4.5% (n=4) becomes the insulin positive after 48 hours, shown in Fig. 9 B during as colored printing.
Compare with the cell that BTC handles with activin A, activin A is very different with the form of the cell that BTC-δ 4 handles.Some insulin positive cells demonstrate the process of prolongation, but major part looks that maintenance is circular.The nucleus of these cells is shrinkages, is fragmentation in some cases or does not exist, and shown in Fig. 9 B and D during as colored printing, this is the feature that cell passes through apoptosis death.Consistent with the apoptosis phenomenon, many cells of handling with activin A and BTC-δ 4 are TUNEL-positives, and only have sub-fraction to become the TUNEL-positive with the cell that activin A and BTC handle, as shown in figure 10.
In order to reaffirm the influence of 4 pairs of AR42J-B20 cell survivals of BTC-δ, we have measured the change that activin A and BTC or BTC-δ 4 handle the back viable count.After 48 hours, the viable count in the culture of handling with activin A and BTC be inoculation cell number 78.8 ± 4.2%.Compare, with the viable count obviously lower (21.8 ± 3.2%) (P＜0.005) in the culture of activin A and BTC-δ 4 processing.In a word, BTC-δ 4 is effective equally with BTC aspect stimulation AR42J-B20 cell differentiation; Yet the effect of BTC-δ 4 aspect these cell survivals of promotion is much smaller.
Embodiment 7: The rat that BTC-δ 4 is given the STZ-processing can reduce plasma glucose concentration and improve Fructus Vitis viniferae The sugar tolerance
For beta cell differentiation activity in the body of studying BTC-δ 4, we give the neonate rat that STZ-handles with BTC-δ 4.Testing program is by the animal care committee approval of Gunma university.With fresh Sprague-Dawley (SD) rat that is dissolved in 85 μ g/g streptozotocin (STZ) intraperitoneal (ip) injection, one age in days of 0.05mmol/l citrate buffer (pH 4.5).Cub is stayed mother at one's side up to 4 ages in week.(the 1st day) tests the blood glucose of all new born animal with Accu-Chek Active (Roche Diagnostics, Germany) after STZ injects 1 day.Have only when first day blood sugar concentration 200 and 350mg/dl between the time, just this animal is included in this research.Serious diabetes take place in the animal of first day blood glucose＞350mg/dl, at blood sugar concentration＞600mg/dl of the 2nd day or the 3rd day these animals.
3pmol/g BTC-δ 4, BTC or saline are injected the neonate rat of handling to STZ-every day, amounted to 5 days from the 0th day.Measure fasting serum glucose concentration and body weight in first every day in week, measure once weekly then, measured for 8 weeks altogether.STZ handled back 2 months, and ip glucose tolerance test (2g/kg body weight) is carried out in fasting after 14 hours.
The 4th day and the 8th week,, put to death after 3 hours with every 100g body weight 1ml bromodeoxyribouridine (BrdU) labelled reagent ip injection animal (cell proliferation reagent box, Amersham Pharmacia Biotech, Britain).The excision pancreas is weighed, and is divided into two parts.With the fixing a spleen sections (4 ℃ are spent the night) of 4% paraformaldehyde/PBS, be used for paraffin embedding after the processing.On each new born animal pancreas, cut 4 sections continuously with the interval of 100 μ m, adults be 300 μ m at interval, carry out immunostaining and histochemistry.Homogenate second portion in cold acid-ethanol, heating is 5 minutes in 70 ℃ of water-baths, and is centrifugal, supernatant is stored in-20 ℃, measures insulin then.Measure insulin by time-resolved immunofluorescence test as previously mentioned (Mashima etc., 1996).
(Li etc., 2001 as previously mentioned; Li etc., 2003) carry out SABC.The source of first antibody and dilute as follows: Cavia porcellus is anti--Iletin II (Lilly), 1: 1000 (doctor Matozaki of Gunma university provides); Rabbit resists-PDX-1,1: 3000; Monoclonal mouse anti-BrdU, 1: 100 (Amersham); The rabbit that is used for the wide spectrum screening resists-Niu cytokeratin (Ckwss) 1: 1000 (DAKO).The source of second antibody and dilute as follows: goat Alexa Flour 568 link coupled anti--Cavia porcellus IgG, 1: 1000; Goat Alexa Flour 488 link coupled anti--Cavia porcellus IgG, 1: 500; Goat Alexa Flour 568 link coupled anti--mice IgG, 1: 1000; Goat Alexa Flour 488 link coupled anti--mice IgG, 1: 500; Goat Alexa Flour 568 link coupled anti--rabbit igg, 1: 1000; Goat Alexa Flour 488 link coupled anti--rabbit igg, 1: 500 (Molecular Probes Inc., Oregon Eugene).
By the PXL 1400 (Photometrics of cooling-charge-coupled device camera system are housed, the State of Arizona, US Tucson) AX70 epifluorescence microscope (Olympus, the Tokyo) the beta cell quality is carried out quantitatively in insulin-painted section with image analysis software (NIH image), described camera arrangement is to operate with IP Lab Spectrum software (Signal Analysis, Vienna, Virginia).In a section (from three sections of the different serial section of each embedded block), measure the insulin-positive cell zone under 40 visuals field (amplification * 200) at least at random.(Li L, Seno M, Yamada H, Kojima I as described elsewhere, the β tunicin can promote beta cell regeneration (Promotion of β-cell regeneration by betacellulin inninety percent pancreatectomized rats) in the rat of 90% pancreas excision, Endocrinology 142:5379-5385,2001; Li L, Seno M, Yamada H, Kojima I: the β tunicin improves glucose metabolism (Betacellulin improves glucose metabolismby promoting conversion of Intraislet precursor cells to β-cells in streptozotocin-treatedmice) by promoting that precursor is converted into beta cell in the island in the mice of streptozotocin-processing, Am J Physiol 285:E577-E583,2003).For quantitative beta cell new life, we have measured the quantity (width is less than 5 cells) of island like cell bunch (ICC).Measure quantity, measure the area of these sections with ICC and island in the painted section of anti-insulin antibody.With regard to each animal, analyze 5 sections at least.The result is expressed as meansigma methods ± SE, sees Table 1 and Figure 11 and 12 (during Figure 12 colored printing).In the comparison between two groups, adopt not t-check in pairs.
Table 1.
Give the feature of the neonate rat that the STZ-of BTC or BTC-δ 4 handles.
Normally STZ STZ/BTCδ4 STZ/BTC
Heavy (g) plasma glucose (mg/dl) plasma insulin (ng/ml) insulin content (μ g) the B-cell quality (mg) of body weight (g) pancreas 224±12.0(6) 0.80±0.03(6) 131.5±6.0(6) 2.8±0.5(6) 105.9±5.2(6) 8.06±0.70(6) 212±9.9(5) 0.82±0.06(5) 160.0±6.2(5) 0.99±0.3(5) 39.0±5.4(5) 2.40±1.0(5) 21 9±8.4(5) 0.91±0.08(5) 139.2±3.4(5) ** 1.92±0.4(5) * 70.3±5.5(5) ** 5.41±0.8(5) ** 220±8.1(5) 0.92±0.06(5) 150.1±5.3(5) * 1.14±0.3(5) 52.4±5.5(5) * 4.12±0.9(5) *
Data are meansigma methods ± S.E. (n).With the neonate rat that BTC, BTC-δ 4 or saline treatment STZ-handle, the 8th week was measured various parameters.
*P＜0.05 to the STZ group. *P＜0.01 to the STZ group.
In the neonate rat that STZ-handles, plasma glucose concentration significantly increases, and reached peak value (＞400mg/dl) (Figure 11 A) reduced gradually then, but still be significantly higher than control rats (table 1) after 2 months at second day.On the contrary, after the rat that STZ-handles gave BTC-δ 4, significantly lower at the 1st day plasma glucose concentration, peak value reduced (Figure 11 A) greatly.Plasma glucose concentration also reduces subsequently, after 2 months (table 1).Compare with the effect of BTC-δ 4, BTC improves the ability relatively poor (Figure 11 A, table 1) of hyperglycemia.When 2 monthly ages, carry out intraperitoneal (ip) glucose tolerance test.In the rat that STZ-handles, significantly impaired to the glucose response that ip glucose-loading produces, however in the rat that the STZ-that gives BTC-δ 4 handles, glucose response significantly improves (Figure 11 B).BTC-δ 4 has also improved insulin response, but compares with normal rat, and the insulin response that ip glucose-loading is produced still is delayed.In the rat that BTC-handles, improved the glucose tolerance, but the effect of BTC is less than BTC-δ 4 (Figure 11 B).And, insulin content and beta cell quality significantly increase (table 1) in the rat that BTC-δ 4-handles, show that at the 4th day histologic analysis BTC-δ 4 has significantly increased the quantity (Figure 12 is during colored printing) of positive vessel cell of PDX-1-and ICC to the pancreas tissue.
Those skilled in the art obviously understand, though describe the present invention in detail for the purpose of setting forth and understand, can carry out various modifications and change to embodiment as herein described and method under the situation that does not deviate from the disclosed inventive concept scope of this description.
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Sequence table
＜110〉Gropep Ltd. (Gropep Limited)
＜120〉method of treatment diabetes
<130>VS：AJH：FP20106
<160>15
<170>PatentIn version 3.3
<210>1
<211>20
<212>DNA
＜213〉synthetic
<400>1
gagcggggtt gatggaccgg 20
<210>2
<211>21
<212>DNA
＜213〉synthetic
<400>2
ttaagcaata tttgtctctt c 21
<210>3
<211>29
<212>DNA
＜213〉synthetic
<400>3
ctcgtcgacg agcggggttg atggaccgg 29
<210>4
<211>30
<212>DNA
＜213〉synthetic
<400>4
ctcctgcagt taagcaatat ttgtctcttc 30
<210>5
<211>32
<212>DNA
＜213〉synthetic
<400>5
cgtccatggc tgatgggaat tccaccagaa gt 32
<210>6
<211>33
<212>DNA
＜213〉synthetic
<400>6
cgtctcgagt cattaagcaa tatttgtctc ttc 33
<210>7
<211>32
<212>DNA
＜213〉synthetic
<400>7
cgtccatggc tgatgggaat tccaccagaa gt 32
<210>8
<211>30
<212>DNA
＜213〉synthetic
<400>8
cgtctcgagt cagtaaaaca agtcaactgt 30
<210>9
<211>51
<212>DNA
＜213〉synthetic
<400>9
ctcgggaatt ccgactacaa ggacgacgat gacaagacca gaagtcctga a 51
<210>10
<211>30
<212>DNA
＜213〉synthetic
<400>10
ctcctgcagt taagcaatat ttgtctcttc 30
<210>11
<211>129
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>11
Met Asp Arg Ala Ala Arg Cys Ser Gly Ala Ser Ser Leu Pro Leu Leu
1 5 10 15
Leu Ala Leu Ala Leu Gly Leu Val Ile Leu His Cys Val Val Ala Asp
20 25 30
Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp
35 40 45
Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly
50 55 60
His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly
65 70 75 80
Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Pro Leu
85 90 95
Arg Lys Arg Arg Lys Arg Lys Lys Lys Glu Glu Glu Met Glu Thr Leu
100 105 110
Gly Lys Asp Ile Thr Pro Ile Asn Glu Asp Ile Glu Glu Thr Asn Ile
115 120 125
Ala
<210>12
<211>94
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>12
Met Asp Arg Ala Ala Arg Cys Ser Gly Ala Ser Ser Leu Pro Leu Leu
1 5 10 15
Leu Ala Leu Ala Leu Gly Leu Val Ile Leu His Cys Val Val Ala Asp
20 25 30
Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly Asp
35 40 45
Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys Gly
50 55 60
His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys Gly
65 70 75 80
Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val
85 90
<210>13
<211>63
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>13
Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly
1 5 10 15
Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys
20 25 30
Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys
35 40 45
Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val
50 55 60
<210>14
<211>98
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>14
Asp Gly Asn Ser Thr Arg Ser Pro Glu Thr Asn Gly Leu Leu Cys Gly
1 5 10 15
Asp Pro Glu Glu Asn Cys Ala Ala Thr Thr Thr Gln Ser Lys Arg Lys
20 25 30
Gly His Phe Ser Arg Cys Pro Lys Gln Tyr Lys His Tyr Cys Ile Lys
35 40 45
Gly Arg Cys Arg Phe Val Val Ala Glu Gln Thr Pro Ser Cys Val Pro
50 55 60
Leu Arg Lys Arg Arg Lys Arg Lys Lys Lys Glu Glu Glu Met Glu Thr
65 70 75 80
Leu Gly Lys Asp Ile Thr Pro Ile Asn Glu Asp Ile Glu Glu Thr Asn
85 90 95
Ile Ala
<210>15
<211>35
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>15
Pro Leu Arg Lys Arg Arg Lys Arg Lys Lys Lys Glu Glu Glu Met Glu
1 5 10 15
Thr Leu Gly Lys Asp Ile Thr Pro Ile Asn Glu Asp Ile Glu Glu Thr
20 25 30
Asn Ile Ala
35

Claims (26)

1. suffering from diabetes or be in treatment in the object of suffering from diabetes risk, prevent this disease or postpone the method for its morbidity for one kind, described method comprises and gives described object polypeptide, this polypeptide can stimulate island cell CFU-GM to be divided into pancreatic beta cell, and compare with real BTC, this polypeptide does not have cell growth-promoting activity or this active reduction.
2. suffering from diabetes or be in treatment in the object of suffering from diabetes risk, prevent this disease or postpone the method for its morbidity for one kind, described method comprises and gives described object nucleic acid, this nucleic acid coding can stimulate island cell CFU-GM to be divided into the polypeptide of pancreatic beta cell, compare with real BTC, described polypeptide does not have cell growth-promoting activity or this active reduction.
3. the polypeptide that can stimulate island cell CFU-GM to be divided into pancreatic beta cell is treated, is prevented this disease or postpones the application of its morbidity in diabetics, wherein compare with real BTC, and described polypeptide does not have cell growth-promoting activity or this active reduction.
4. the coding nucleic acid that can stimulate island cell CFU-GM to be divided into the polypeptide of pancreatic beta cell is suffering from diabetes or is being in treatment in the object of suffering from diabetes risk, prevents this disease or postpones the application of its morbidity, wherein compare with real BTC, described polypeptide does not have cell growth-promoting activity or this active reduction.
5. can stimulate polypeptide that island cell CFU-GM is divided into pancreatic beta cell being used for suffering from diabetes or being in the object treatment of suffering from diabetes risk, preventing this disease or postponing application in the medication preparation of its morbidity, wherein compare with real BTC, described polypeptide does not have cell growth-promoting activity or this active reduction.
6. the coding nucleic acid that can stimulate island cell CFU-GM to be divided into the polypeptide of pancreatic beta cell is being used for suffering from diabetes or is being in the object treatment of suffering from diabetes risk, prevents this disease or postpones application in the medication preparation of its morbidity, wherein compare with real BTC, described polypeptide does not have cell growth-promoting activity or this active reduction.
7. as each described method or application among the claim 1-6, it is characterized in that compare with real BTC, described polypeptide activates the ErbB1 of described object and the ability of ErbB4 receptor significantly reduces.
8. as each described method or application among the claim 1-7, it is characterized in that, stimulate island cell CFU-GM not stimulate the propagation of other cell type to the pancreatic beta cell differentiation phase.
9. as each described method or application among the claim 1-8, it is characterized in that described diabetes are type 2 diabetes mellitus.
10. as each described method or application among the claim 1-8, it is characterized in that described diabetes are type 1 diabetes.
11. a treatment, prevent diabetes or postpone the compositions of its morbidity, it comprises the polypeptide and the pharmaceutically acceptable carrier that can stimulate island cell CFU-GM to be divided into pancreatic beta cell, wherein compare with real BTC, described polypeptide does not have cell growth-promoting activity or this active reduction.
12. a treatment, prevent diabetes or postpone the compositions of its morbidity, it comprises coding can stimulate island cell CFU-GM to be divided into the nucleic acid and the pharmaceutically acceptable carrier of the polypeptide of pancreatic beta cell, wherein compare with real BTC, described polypeptide does not have cell growth-promoting activity or this active reduction.
13., it is characterized in that as each described method, application or compositions among the claim 1-12, to compare with real BTC, described polypeptide does not have the ability of irritation cell type propagation or this ability to reduce.
14., it is characterized in that as each described method, application or compositions among the claim 1-13, to compare with real BTC, ability or this ability that described polypeptide does not activate ErbB1 or ErbB4 receptor reduce.
15., it is characterized in that as each described method, application or compositions among the claim 1-14, to compare with real BTC, ability or this ability that described polypeptide does not activate ErbB2 or ErbB3 homodimer reduce.
16., it is characterized in that compare with the risk that produces with real BTC treatment, the risk that cancer takes place in object as the side effect for the treatment of reduces as each described method, application or compositions among the claim 1-15.
17., it is characterized in that described polypeptide and the basic homology of EGF family member as each described method, application or compositions among the claim 1-16.
18. method as claimed in claim 17, application or compositions, it is characterized in that the basic homology of described polypeptide and epidermal growth factor, transforminggrowthfactor-, heparin-bounding EGF like growth factor, epiregulin, amphiregulin, nerve and thymus-derived ErbB kinase activator thing (NTAK), neuregulin subfamily member (NRG1, NRG2, NRG3 and NRG4) or β tunicin (BTC).
19. method as claimed in claim 18, application or compositions is characterized in that, described polypeptide and BTC-δ 4 basic homologies.
20. method as claimed in claim 19, application or compositions is characterized in that, described polypeptide is BTC-δ 4.
21. method as claimed in claim 20, application or compositions is characterized in that, described BTC-δ 4 polypeptide have aminoacid sequence shown in Figure 4 (SEQ ID NO:11).
22., it is characterized in that described polypeptide is as each described method, application or compositions among the claim 1-12:
(a) splice variant of BTC does not wherein have C 5-C 6Two sulfur rings, and have this two sulfur ring in the gene of the real BTC that encodes usually; Or
(b) sequence and (a) homologous substantially polypeptide, or
(c) analog (a), fragment, mutant, derivant or allele variant.
23., it is characterized in that described polypeptide is as each described method, application or compositions among the claim 1-12:
(a) splice variant of BTC does not wherein have C 5-C 6Two sulfur rings, and have this two sulfur ring in the gene of the real BTC that encodes usually, the remainder of ripe molecule comprises that A ring and B ring and " hinge " valine frame endomixis are in the terminal kytoplasm tail of the C-of truncate;
(b) sequence and (a) homologous substantially polypeptide; Or
(c) fragment (a), analog, variant or derivant.
24. as each described method, application or compositions among the claim 1-23, it is characterized in that described polypeptide can be used as the beta cell differentiation factor, increases the beta cell quality, reduce the reduction of beta cell function and/or the insulin secretion of increase beta cell.
25., it is characterized in that compare with the risk that produces with the BTC treatment, the risk that cancer takes place in object as the side effect for the treatment of reduces as each described method, application or compositions among the claim 1-24.
26. as each described method, application or compositions among the claim 1-25, it is characterized in that described object has in the following diabetes risk factor one or multinomial: the glucose tolerance is impaired, metabolism syndrome, 1 type or type 2 diabetes mellitus family history, obesity, polycystic ovarian syndrome, hypertension or hypercholesterolemia.
CNA2005800324194A 2004-08-06 2005-08-05 Method for treating diabetes Pending CN101090732A (en)

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CN110279860A (en) * 2019-07-31 2019-09-27 上海交通大学医学院附属瑞金医院 Nrg4 is preparing the application in diabetes medicament as target spot
CN114075272A (en) * 2020-08-10 2022-02-22 杭州俊丰生物工程有限公司 Preparation method of human neuregulin 4
CN114796292A (en) * 2013-03-12 2022-07-29 豪斯制药研究实验室有限责任公司 Plant extracts with anti-diabetic and other useful activities

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CU23526B6 (en) 2006-10-03 2010-05-19 Ct Ingenieria Genetica Biotech METHOD FOR MORFOFUNCTIONAL RESTORATION OF PERIPHERAL NERVES IN DIABETIC NEUROPATHY
KR102027750B1 (en) * 2018-05-03 2019-10-02 연세대학교 산학협력단 Composition for emitting glucose
WO2021002645A1 (en) * 2019-07-01 2021-01-07 연세대학교 산학협력단 Composition for excretion of sugar comprising hydrogel and egfr ligand as active ingredients
KR102109385B1 (en) * 2019-08-21 2020-05-12 연세대학교 산학협력단 Composition for emitting glucose

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AUPQ496900A0 (en) * 2000-01-06 2000-02-03 Gropep Pty Ltd Growth factor splice variant

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CN114796292A (en) * 2013-03-12 2022-07-29 豪斯制药研究实验室有限责任公司 Plant extracts with anti-diabetic and other useful activities
CN110279860A (en) * 2019-07-31 2019-09-27 上海交通大学医学院附属瑞金医院 Nrg4 is preparing the application in diabetes medicament as target spot
CN114075272A (en) * 2020-08-10 2022-02-22 杭州俊丰生物工程有限公司 Preparation method of human neuregulin 4
CN114075272B (en) * 2020-08-10 2023-09-22 杭州俊丰生物工程有限公司 Preparation method of human neuregulin 4

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