WO2007009356A1 - A method for detecting and culturing pancreatic cells and their application - Google Patents

A method for detecting and culturing pancreatic cells and their application Download PDF

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WO2007009356A1
WO2007009356A1 PCT/CN2006/001669 CN2006001669W WO2007009356A1 WO 2007009356 A1 WO2007009356 A1 WO 2007009356A1 CN 2006001669 W CN2006001669 W CN 2006001669W WO 2007009356 A1 WO2007009356 A1 WO 2007009356A1
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streptomycin
penicillin
medium
dmem
pancreatic
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French (fr)
Chinese (zh)
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Yu Zhang
Chunlin Zou
Biao Chen
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Theracells Biotechnologies Co., Ltd.
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0678—Stem cells; Progenitor cells; Precursor cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/507—Pancreatic cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5073—Stem cells
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    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/335—Glucagon; Glucagon-like peptide [GLP]; Exendin

Definitions

  • the present invention relates to stem cells, and more particularly to culturing and expanding islet stem cells from pancreatic islet cell populations isolated from pancreatic tissues of diabetic animals (specially ij is a P-milk animal), and further diluting them into pancreatic endocrine cells. method. BACKGROUND OF THE INVENTION Since the first successful isolation and cultivation of human embryonic stem cells by James Thomson in 1998, since stem cells have important application prospects for the treatment of future diseases, stem cell technology has undoubtedly become one of the research hotspots in the field of biotechnology. At present, there are more than 30 million diabetic patients in China, and they are increasing at a rate of 750,000 new patients per year.
  • Stem cells are a kind of cells that can self-replicate, self-renew and have the potential to differentiate into the corresponding tissue cells. Therefore, it has become a research focus to find a stem cell that can be expanded in vitro and can differentiate into islet ⁇ cells.
  • stem cells which can differentiate into islet ⁇ cells in vitro mainly have the following three types: embryonic stem cells (ES), pancreatic duct-derived stem cells, and islet-derived stem cells.
  • ES embryonic stem cells
  • pancreatic duct-derived stem cells pancreatic duct-derived stem cells
  • islet-derived stem cells Details of the research are in Shi, Y., Hou, L., Tang, F., Jiang : W., Wang, P., Ding, M., & Deng, H. ( 2005 ) .
  • Stem Cells, 2005, 23, 656-662. Bonner-Weir, S., Taneja, M., Weir, G. C, Tatarkiewicz, ., Song, KH, Sharma, A., &O'Neil, JJ
  • Lumelsky et al. 4 through the in vitro five-step induction method, can induce embryonic stem cells to differentiate into islet ⁇ cells, and Shi et al. in their study 4, they passed three steps The method can induce embryonic cells into islet ⁇ cells, and transplanting these cells into diabetic mice can increase the body weight, prolong the survival time, and lower blood sugar.
  • the method can induce embryonic cells into islet ⁇ cells, and transplanting these cells into diabetic mice can increase the body weight, prolong the survival time, and lower blood sugar.
  • due to the tumorigenicity problems that are still unsolvable in embryonic stem cells there are still a few major gaps away from clinical use.
  • Bonner-Weir et al. reported in their study that there is a CK19 P-stem stem cell in the pancreatic duct.
  • Such cells can be expanded and differentiated into islet ⁇ fine packets in the presence of KGF and Matrigel.
  • Zulewski et al. reported that they isolated a cell expressing the neural stem cell marker nestin (nestin) from the pancreatic islets of the normal pancreas, which can be expanded in vitro and can be The effects of exendin-4, activin A, HGF, Betacellulin and nicotiamide differentiate into pancreatic endocrine endocrine packets.
  • Nestin is a high molecular weight intermediate filament protein. Nestin is expressed in fibroblasts in differentiated cells, but not in epithelial cells, but its expression is observed in undifferentiated ancestral epithelial cells by expressing nestin.
  • Pancreatic duct undifferentiated epithelioid cells induce differentiation into a subset of islet cells, demonstrating that nestin is a molecular marker of pancreatic stem cells, which may play a role in promoting pancreatic endocrine dry fine-package differentiation.
  • nestin is a molecular marker of pancreatic stem cells, which may play a role in promoting pancreatic endocrine dry fine-package differentiation.
  • the dried pancreas J! package is an islet-derived adult stem cell.
  • the tissue used by Zulewski et al. is normal human or murine pancreatic tissue. Since it is difficult to obtain a sufficient amount of normal human pancreatic tissue in practice, the application of this technique to clinical practice still faces problems of insufficient source. Therefore, it is necessary to solve the problem of insufficient source of pancreatic tissue in islet transplantation, and it is also necessary to solve the toxicity problem caused by the use of immunosuppressive agents in allogeneic transplantation.
  • the main object of the present invention is to use pancreatic stem cells remaining in a diabetic animal to be isolated and cultured in vitro, and then differentiate into functional mature islet ⁇ cells under the action of an inducing factor, and then transplanted into the pancreatic islet ⁇ cells. In animals or other animals.
  • This method of autografting not only solves the problem of insufficient source of pancreatic tissue in islet transplantation, but also solves the toxicity problem caused by the use of immunosuppressants in allogeneic transplantation.
  • a method for detecting the presence or absence of pancreatic stem cells in pancreatic tissue of a diabetic animal comprising: a) immunohistochemical detection, comprising: pancreatic tissue fixation, preparation of paraffin embedded sections, and sealing after exposure Anti-antibody, contacted with secondary antibody, and then blocked, wherein the primary antibody comprises a rabbit anti-nestin antibody; b) RT-PCR analysis, including extraction of total RNA, reverse transcription to form cDNA, and then PCR amplification using primers c) determining the presence or absence of pancreatic stem cells in the pancreatic tissue of diabetic animals based on the results of steps a) and b).
  • a method for isolating residual islet cell mass from a diabetic animal pancreatic tissue comprising the steps of: a) cutting the pancreatic tissue into pieces; b) The pancreatic tissue is subjected to enzymatic digestion, and then the digestion is terminated; c) centrifugation, de-clearing, washing; d) obtaining isolated islet cell clusters.
  • a method for isolating and cultivating islet stem cells comprising the steps of: a) adding islet cell clusters isolated from pancreatic tissue of a diabetic animal to a culture medium containing medium A, Incubate for 2-5 days; b) inoculate suspended islet cells in culture medium containing medium B; c) N2006/001669 Astrocytic stem cells are grown after 6-20 days; d) Pancreatic stem cells are obtained by passage when the star islet stem cells are confluent.
  • the medium A includes: a medium, a buffer, a serum, an amino acid, an antibiotic, and the like.
  • the basal medium includes RPMI 1640 or the like; the serum includes FBS or the like; the buffer includes 4-hydroxyethylpiperazine ethyl sulfonate buffer, sodium pyruvate buffer, etc.; and the amino acid includes L- Glutamate or the like; the antibiotic includes penicillin-streptomycin and the like.
  • An example of medium A is: RPMI 1640, 8-15% FBS, 10 mM 4-hydroxyethyl, Qin E lateral buffer, 1 mM sodium pyruvate buffer, 2 mM L-glutamic acid, lOOu penicillin-lOOug Streptomycin; another example is: RPMI 1640, FBS, HEPES (100X), acetone oxime] (100X), L-glutamine (100X), Antibiotic (100 ⁇ ).
  • the skilled person or the skilled person can make some changes or modifications to the composition and quantity of the medium A according to the common sense and the specific conditions of the present, for example, the basic medium can be DMEM.
  • the serum may be horse serum, newborn calf serum, and the buffer may be sodium bicarbonate buffer, and all such changes and modifications are included in the scope of the present invention.
  • the medium B includes: a basal medium, a buffer, serum, an amino acid, an antibiotic, and a growth factor.
  • the basal medium includes RPMI 1640 or the like; the serum includes FBS or the like; the buffer includes 4-hydroxyethyl piperazine B transverse buffer, sodium pyruvate buffer, etc.; the amino acid includes L-Valley
  • the antibiotic includes penicillin-streptomycin or the like; the growth factor includes basic fibroblast growth factor, epidermal growth factor, and the like.
  • An example of medium B is: RPMI 1640, 8-15% FBS, 10 mM 4-hydroxyethyl pipe "QIN Etra buffer, 1 mM sodium pyruvate buffer, 2 mM L-glutamate, lOOu penicillin - 100ug streptomycin, 10-20ng/ml basic fibroblast growth factor, 10- 20ng/ml epidermal growth factor; another example: RPMI 1640, FBS, HEPES (100X), sodium acetonide, L- Glutamine (100X), Antibiotic (100X), Human bFGF, Human EGF.
  • the basic medium can be DMEM
  • the serum can be horse serum, newborn cattle (fetal calf) serum
  • the buffer can be sodium bicarbonate buffer, all these changes and modifications It should be included in the scope of the invention.
  • PPC pancreatic islet stem cells
  • GLP-1 glucagon-like growth factor
  • HGF hepatic serotonin growth factor
  • betacellulin betacellulin
  • nicotinamide nicotinamide
  • a method for culturing islet stem cells into endocrine cells of a melanogaster gland comprises the following steps: a) inoculation of islet stem cells in a culture dish containing medium C for about 2-5 days; b) replacing medium C with Medium D, but still in culture, cultured for about 4-15 days to obtain a secretory pericardium that can secrete insulin.
  • the culture subculture is a bacterial culture J ⁇ or; a cell culture sub-containing 0.01% polyornithine or polylysine.
  • the medium C comprises: a basic medium, a culture additive, an albumin, an antibiotic, and a growth factor.
  • the basal medium includes DMEM and Nutrient Mixture Ham's F-12.
  • the culture additive includes B27 or (insulin + transferrin + sodium selenite) ITS; the albumin includes BSA; the antibiotic includes penicillin-streptomycin; and the growth factor includes basic fibril fine Growth factor, epidermal growth factor.
  • An example of the medium C is: 48% DMEM, 48% Nutrient Mixture Ham's F-12 ⁇ 2% B27 or lg/L ITS (ie 5 mg/L insulin + 5 mg/ L transferrin + 5ml / L sodium selenite), 0.05% - 0.2% BSA, lOOu penicillin - lOOug streptomycin, 10- 20ng / ml basic fibroblast growth factor, 10-20ng / ml epidermal cell growth Factor;
  • An example is: DMEM/F12 l: l (8 mM glucose), B27 (50X, GIBCO), 0.075% BSA, bFGF 20 ng/ml, EGF 20 ng/ml, Antibiotics (100X, GIBCO)'.
  • the medium D includes: a basal medium, a culture additive, albumin, an antibiotic, a polypeptide having an i-show function, and a growth factor.
  • the basal medium comprises DMEM and Nutrient Mixture Ham's F-12, and the culture addition agent comprises B27 or ITS (ie, insulin + transferrin + sodium selenite); the albumin comprises BSA;
  • Antibiotics include penicillin-streptomycin;
  • the polypeptides and growth factors include: nicotinamide, GLP-HGF, Betacellulin.
  • An example of such medium D is: 48% DMEM, 48% Nutrient Mixture Ham's F-12, 2% B27 or lg/L ITS (ie 5 mg/L insulin + 5 mg/L transferrin + 5 mg/L sub- Sodium selenate), 0.05%-0.1% BSA, lOOu penicillin-lOOug streptomycin, lOmM nicotinamide, 10-lOOnM GLP-1, lOng/ml HGF, 500 pmol/L Betacellulin; another example: DMEM/F12 1:1 (5.6 mM glucose), B27 (50X, GIBCO), 0.075% BSA, lOmM nicotinamide, Antibiotics (100X, GIBCO), GLP-1 (7-36amide), HGF, Betacellulin.
  • an islet cell mass obtained by the aforementioned method for isolating islet cell mass from pancreatic tissue of a diabetic animal.
  • an islet stem cell obtained by the method for isolating and culturing the islet stem cells described above.
  • Figure 1B shows immunohistochemical results (a, c, e) of islets in normal monkey pancreatic tissue and immunohistochemical results (b, d, f) of islets in diabetic pancreatic tissue.
  • a and b represent the islet ⁇ fine packets of normal monkeys and diabetic monkeys, respectively;
  • c and d represent the glucagon cells ( ⁇ cells) of normal monkeys and diabetic monkeys, respectively;
  • e and f represent normal monkeys and diabetes, respectively.
  • the delta cell condition of the monkey The results showed that diabetic monkey islets (3 cells were almost completely destroyed (b and a), glucagon cells increased significantly, and ⁇ cells secreting somatostatin did not change significantly.
  • Figure 1C shows immunohistochemistry results for Nestin gene, a is the result of normal monkeys, and b is the result of diabetic monkeys.
  • Figure 1C shows that for normal monkeys and diabetic monkeys, there is Nestin expression in pancreatic tissue, but the distribution of Nestin-positive cells varies, in pancreatic tissue of normal monkeys.
  • Nestin-positive cells are distributed around the glandular gland and islet clusters, while in the pancreatic tissue of diabetic monkeys, Nestin P-sex cells are mainly distributed in islets and the number is significantly increased.
  • Figure 2A, 2B, 2C, 2D The results of culture of islets, pancreatic precursor cells, and islet-like cell clusters are shown.
  • Figure 2A shows floating circular or elliptical islet fines in a Petri dish
  • Figure 2B shows that The isolated nestin-positive islet cell mass was added to medium A, and inoculated in a Petri dish for about 3 days
  • Fig. 2C shows the results of inoculation of the islet cell mass shown in Fig. 2B in ordinary cell culture containing medium B for about 15 days
  • Fig. 2D shows the obtained star islet stem cells
  • FIG. 3A shows the RT-PCR results of pre-pancreatic sputum cells (PPC) and ICC.
  • Figure 3B shows pancreatic precursor cells, which are nestin-positive fine packets.
  • Figure 3C shows that insulin-positive cells in ICC are mainly distributed in ICC. Central and glucagon-positive cells are mainly distributed around the periphery of ICC.
  • Figures 4A and 4B show the secretion of prostaglandins and C-peptides by Pre-ICCs, ICCs, and normal islets stimulated by 30 mM high glucose. The secretion of insulin is shown, and Figure 4B shows the secretion of C-peptide.
  • Figures 5A-5D show photographs of islet cell mass obtained after digestion and culture of pancreatic tissue cells in type V collagenase, wherein Figure 5A shows digestion 5 Minutes, 2 days of results; Figure 5B shows 5 minutes of digestion Figure 5C shows the results of digestion for 10 minutes and culture for 2 days; Figure 5D shows the results of digestion for 10 minutes and culture for 5 days.
  • Figures 6A-6D show the use of medium C in accordance with Example 4 Culture medium D cultures the islet-like structure obtained in JUL and the photo of the pancreatic endocrine cells.
  • FIG. 6A shows an islet-like structure obtained by culturing in medium C for 2 days
  • Fig. 6B shows an islet-like structure obtained by culturing in medium C for 5 days
  • Fig. 6C shows pancreatic endocrine cells obtained by culturing for 10 days in the medium D
  • Fig. 6D shows pancreatic endocrine cells obtained by culturing for 15 days in the medium D.
  • Figure ⁇ shows the RT-PCR results of normal and diabetic animal pancreatic spring tissues.
  • Figure 8 shows the RT-PCR results of PPC and ICC.
  • Figure 9 shows the secretion of insulin and C-peptide when ICC and adult islets are stimulated with different concentrations of glucose or with 10 mM L-arginine, wherein Figure 9A1 shows ICC in the presence of different concentrations of glucose or glucose. Insulin secretion in the presence of 10 mM L-arginine; Figure 9A2 shows insulin secretion in the presence of different concentrations of glucose or glucose and 10 mM L-arginine in adult islets; Figure 9B 1 Shows the secretion of C-peptide in the presence of ICC in the presence of different concentrations of glucose or glucose and 10 mM lysine; Figure 9B2 shows the presence of adult islets in the presence of different concentrations of glucose or glucose and 10 mM L-arginine The secretion of the lower C-peptide.
  • insulin indicates the insulin gene
  • Glucagon indicates the glucagon gene
  • Somatostatin indicates the somatostatin gene
  • PDX-1 indicates the knee duodenal homeobox gene
  • nestin indicates the nestin gene
  • ⁇ -actin indicates ⁇ -actin gene
  • "-" means normal monkey
  • + STZ means diabetic monkey
  • Glut-2 means glucose transporter-2 gene
  • Pre-ICC means ICC before entering medium D
  • ICC means cultured ICC after differentiation of base D
  • Islet represents islets.
  • pancreatic stem cells For animals with diabetes (mammals such as humans or monkeys), it is believed that it is not possible or difficult to isolate pancreatic stem cells from the pancreas of animals with diabetes because their pancreas does not normally secrete insulin. If pancreatic stem cells are isolated from the normal pancreas of the allogeneic animal, autologous transplantation is not possible, and the problem of rejection remains, and the pancreatic cells isolated from the normal pancreas come. N2006/001669 source issues cannot be resolved. The inventors of the present invention found that nestin-positive pancreatic stem cells still remain in the pancreas of diabetic animals.
  • such cells have been successfully isolated and cultured in vitro, and the cells have the molecular biological characteristics of the following stem cells: positive expression of nestin gene and positive expression of ABCG2 gene, as shown in Fig. 1A-1C Shown.
  • these cells can be expanded for a long period of time, and can differentiate into islet cells under the action of a specific differentiation-inducing factor.
  • ABCG2 is a member of the ABC superfamily. It not only makes tumor cells multi-drug resistant, but also recently found that it plays an important role in maintaining the unique characteristics of a thousand cells (ie, non-differentiation). 4 Barba is also considered as one of the markers of stem cells.
  • the ABCG2 gene expression was positive in pancreatic stem cells obtained according to the method of the present invention.
  • CK19 fine keratin protein 19 (cytokeratin-19, CK-19), Gmyr et ⁇ / found that adult CK19 positive cells can re-express insulin promoter factor 1 in vitro, further indicating that human pancreatic spring pluripotent
  • the presence of a precursor packet also indicates that CK-19 may be one of the molecular markers of pancreatic precursor cells.
  • Pdx-1 is a pancreatic duodenal homeobox gene (pdx-1).
  • the first molecular marker expressed during pancreatic stem cell development is a homologous region protein.
  • PDX-1 plays an important role in the growth and differentiation of pancreas sprouting in the dorsal and ventral side of the intestinal endoderm.
  • the homozygous deletion mutation of pdx-1 causes the pancreas to fail to form.
  • pancreatic stem cells Differentiation of pdx-1 positive embryonic pancreatic stem cells into pancreatic endocrine cells is multi-step, and pdx-1 positive duct-like cells can also produce exocrine acinar cells.
  • the pdx-1 gene expression was negative in pancreatic stem cells obtained according to the method of the present invention.
  • ISL-1 is an important transcription factor that plays an important role in the formation of islet cells.
  • the protein it encodes binds to the potentiation region of the insulin gene. It is expressed in all pancreatic endocrine cells of the adult.
  • the ISL-1 gene expression was negative in pancreatic stem cells obtained according to the method of the present invention.
  • Glut-2 Glucose transporter-2, a specific marker of islet beta cells, is involved in the proliferation of beta cells and the recognition and transport of glucose.
  • the expression of Glut-2 gene was negative in pancreatic stem cells derived from the method of the present invention.
  • Somatostatin is a somatostatin gene.
  • stem cells derived from diabetic animals themselves can be cultured and differentiated in vitro, and can be used for autologous dry-package transplantation treatment of diabetic animals, and can also be used for carrier fine-moon packets in gene therapy of diabetes.
  • the inventors of the present invention found that although ⁇ cells are completely destroyed in the islets of diabetic animals, residual islet cell clusters can still be isolated by digestion of 3 mg/ml type V collagenase, and these islet colonies are attached. It can rapidly grow into a single layer of flat cells, which begin to die in 7-8 days, but at the same time a multi-angled stellate cell begins to proliferate rapidly, and these cells can confluence in about 14-16 days.
  • the results of the cell immunization group showed that these polygonal fine moon packs were nestin-positive fine-moon packets, but the CK19 staining was negative, so the multi-angled stellate cells were islet stem cells (PPC).
  • RT-PCR results of RT-PCR showed that compared with the RT-PCR results of PPC, the expression of nestin gene and ABCG2 gene was weakened or disappeared, and islet related genes began to express, insulin, glucagon, somatostatin and PDX-1, ISL1.
  • Gene expression such as GLUT2 was positive, as shown in Figures 3A-C.
  • the ICC induced by differentiation in the present invention can synthesize insulin and C peptide under the stimulation of 30 mM glucose, and the secretion amount is about 1/8 of that of normal islets, as shown in Figs. 4A and 4B.
  • a method for detecting pancreatic stem cells in pancreatic tissue of a diabetic animal comprises: a) immunohistochemical detection, comprising: pancreatic tissue fixation, making a stone-enriched embedded slice, contacting the primary antibody after the sputum closure, contacting the secondary antibody The antibody is then blocked for observation, wherein the primary antibody comprises a rabbit anti-nestin antibody; b) RT-PCR analysis, including extraction of total RA, formation of cDNA, followed by PCR amplification using primers; c) according to step a) and The results of b) determine the presence of islet stem cells in the pancreatic tissue of diabetic animals.
  • the main hallmark of islet stem cells is nestin.
  • a method for isolating residual islet cell mass from pancreatic tissue of a diabetic animal comprising the steps of: a) cutting the pancreatic tissue into pieces; b) treating the massive pancreas The tissue is subjected to enzymatic digestion, and then the digestion is terminated; c) centrifugation, de-clearing, washing; d) obtaining isolated islet cell clusters.
  • a method for isolating and cultivating islet stem cells comprising the steps of: a) adding islet cells isolated from pancreatic tissue of a diabetic animal to culture i containing medium A, Incubate for 2-5 days; b) inoculate suspended islet cells in culture medium containing medium B; c) After 6-20 days, the star-shaped islet stem cells are grown; d) the islet stem cells are passaged when the star-shaped islet stem cells are confluent, until the subcultured sub-culture cells are obtained, and the islet stem cells are obtained.
  • medium A includes: RPMI 1640 Manufacturer: Invitrogen, Liquenia, USA), 8-15% FBS (Fetal Bovine Serum, fetal bovine serum) (manufacturer: Invitrogen), 10mM HEPES Buffer Solution
  • the medium B includes: RPMI 1640 (manufacturer: Invitrogen), 8-15% FBS (Fetal Bovine Serum, platform beef serum)
  • a method of inducing differentiation of PPC into pancreatic endocrine cells is provided. Combined use of glucagon-like growth factor (GLP-1). Hepatocyte growth factor (HGF), betacellulin, and nicotinamide induce differentiation of PPC into pancreatic endocrine cells.
  • GLP-1 glucagon-like growth factor
  • HGF Hepatocyte growth factor
  • betacellulin betacellulin
  • nicotinamide induce differentiation of PPC into pancreatic endocrine cells.
  • a method for culturing islet stem cells into pancreatic endocrine cells comprising the steps of: a) inoculation of islet stem cells in culture medium containing medium C for 2-5 days; b) replacing medium C with medium D, But still in the cultivation of sub-culture, culture for about 4-15 days, get Insulin-secreting endocrine cells of the meridian gland.
  • the medium C comprises: 48% DMEM (manufacturer: Invitrogen, article number 1 1966), 48% Nutrient Mixture Ham's F-12' (manufacturer: Invitrogen, article number 11765), 2% B27 (manufacturer: Invitrogen) Or 1 g/L (5 mg/L insulin + 5 mg/L transfer+mgyliter selenium) ITS (manufacturer: Sigma, Missouri, USA), 0.
  • BSA bovine serum albumin
  • bFGF Basic Fibroblast Growth Factor
  • EGF Epidermal Growth Factor
  • EGF Epidermal Growth Factor
  • R&D Systems or include: DMEM/F12 1 : 1 ( 8 mM glucose ) , B27 ( 50X , GIBCO ), 0.075% BSA, bFGF 20ng/ml, EGF 20ng/ml, Antibiotics (100X, GIBCO).
  • the medium D includes: 48% DMEM (manufacturer: Invitrogen, article number 11966), 48% Nutrient Mixture Ham's F-12' (corporate: Invitrogen, article number 11765), 2% B27 (manufacturer: Invitrogen) Or (5mg/L insulin +5mg/L transfer+mgyliter selenium) ITS (vendor: Sigma), 0.05%- 0.1% BSA (bovine serum albumin)
  • Hepatocyte growth factor Manufacturer: R&D Systems, Inc., 500pmol/L BetacellulinC manufacturer: R&D Systems
  • DMEM/F12 1 1
  • Example 1 Diabetic animal model Detection of nestin-positive fine-moon pack in pancreatic tissue Obtaining anesthesia of pancreatic tissue of cynomolgus monkey with diabetes: Ketamine 10 mg/kg Atropine 0.04 mg/kg diazepam 1 mg/kg Abdominal skin preparation in the lower abdomen of the xiphoid Incision, incision of the peritoneum into the abdominal cavity, separation of the greater omentum and intestine, a slightly pink pancreas visible at the lower edge of the stomach, the monkey's pancreatic tail is mostly free, a needle is first sewn at the edge of the pancreas, and then wrapped around it A piece of pancreatic tissue approximately 0.5Xlcm in size was sutured, placed in ice-cold Hank's solution containing antibiotics and washed twice. Detection of dried pancreas
  • Antisera used were: rabbit anti-nestin antibody (manufacturer: Chemicon, California, USA), guinea pig anti-insulin antibody (manufacturer: Zymed Laboratories Inc., South Lili, USA), mouse anti-glucagon antibody (manufacturer: Sigma), rabbit anti-somatostatin antibody (DAKO, Denmark).
  • the fluorescent secondary antibodies were: Texas red goat anti-rabbit IgG, Texas red goat anti-mouse Ig G, Texas red goat anti-moon mole IgG, Cy2 goat anti-rabbit IgG, all fluorescent antibodies were diluted 1:200.
  • Rabbit anti-nestin antibody mainly used to detect Nestin protein.
  • Nestin protein is a medium fiber skeleton protein, which has been identified as a marker of neural stem cells in the study of neurodevelopment. At present, most researchers believe that Nestin protein can also be used as a marker for pancreatic stem.
  • Rabbit anti-insulin antibody It is mainly used to detect islet ⁇ cells. Pancreatic islet ⁇ cells are the main functional cells in the endocrine part of the pancreas. It is the only cell that can secrete insulin in the body.
  • Mouse anti-glucagon antibody It is mainly used to detect ⁇ cells in limb islands, and ⁇ cells are cells of islet endocrine glucagon.
  • Rabbit anti-somatostatin antibody mainly used to detect ⁇ fine packets in islets, ⁇ cells are cells of endocrine somatostatin in islets. Results: The immunohistochemical results of the animal model of the present invention showed that the ⁇ cells of the islets were completely destroyed, and the a cells remaining in the islets were obviously proliferated. Nestin-positive cells are normally present at the edge of the islet group and the pancreatic gland. In the model pancreas of the example, nestin-positive cells are mainly present in the islet group, and the distribution in the gland is significantly reduced.
  • FIG. 1B shows immunohistochemical results ( a , c , e) of islets in normal monkey pancreatic tissue and immunohistochemical results (b, d, f) of islets in diabetic pancreatic tissue.
  • the results showed that the islets of diabetic monkeys (all 3 cells were destroyed (b and a), glucagon cells were significantly increased (d and c), and ⁇ cells secreting somatostatin did not change significantly (f and e).
  • the results of immunohistochemistry for the Nestin gene are shown, a is the result of normal monkeys, and b is the result of diabetic monkeys.
  • Figure 1C shows that for normal monkeys and diabetic wolves, the expression of Nestin is found in pancreatic tissue, only the distribution of Nestin-positive cells.
  • RT-PCR analysis method Total RNA of fine moon pack or tissue is extracted by TRIZOL method, 2 g of total RNA is reverse-transcribed into cDNA, and RT-PCR reaction system consists of 50 mM Tris-HCl (pH 8.3), 50 mM KC1, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM spermidine, 20 U of Riiasin, 0.5 mM dNTP, 0.5 ⁇ g of 01igo(dT) 15 , 50 U of SuperscriptTM II RT (Invitrogen), and ribozyme The water was composed, and then the reaction system was subjected to a water bath at 42 ° C for 60 minutes and a water bath at 72 ° C for 15 minutes.
  • the target gene was then amplified by PCR at 30-45 cycles.
  • the PCR reaction system consisted of 10 mM Tris-HCl, 50 mM KC1, 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 ⁇ ⁇ of sense and Antisense primer, 6.25 U Taq DNA polymer 3 ⁇ 4se and 3 g of cDNA.
  • the temperature of '1' was 94 ° C for 45 seconds, the annealing temperature was determined according to the specific primer, the time was 30 seconds, and the extension temperature was 72 ° C for 7 minutes.
  • the PCR product was separated by 1.5% gel electrophoresis. sequence:
  • Insulin upstream primer TCA CAC CTG GTG GAA GCT C (SEQ ID NO: 1)
  • downstream primer ACA ATG CCA CGC TTC TGC (sequence 2) (179 bp).
  • Glucagon upstream primer ATT CAC AGG GCA CAT TCA CC (SEQ ID NO: 3), downstream primer: AAC AAT GGC GAC CTC TTC TG (sequence 4) (260 bp) o
  • Somatostatin upstream primer GTT TCT GCA GAA GTC TGG G (Sequence ⁇ 'J 5 ), downstream primer: AGT TCT TGC AGC CAG CTT TG (Sequence 6) (223 bp).
  • PDX-1 upstream primer GGA TGA AGT CTA CCA AAG CTC ACG C (Sequence ⁇ 'J 7 ), downstream primer: CCA GAT CTT GAT GTG TCT CTC GGT C (SEQ ID 8 ) (218 bp).
  • ABCG2 upstream primer GGT CTC AGG AAG ACT TAT GT (Sequence ll 11 ), downstream primer: AAG GAG GTG GTG TAG CTG AT (Sequence 12) (323 bp).
  • Isll upstream primer CTT AAA TTG GAC TCC TAG AT (SEQ ID NO: 13), downstream primer: GGA TTT GGA ATG GCA TGC GG (SEQ ID NO: 14) (280 bp).
  • insulin gene (Insulin) expression was negative
  • glucagon gene (Glucagon) and somatostatin gene (Somatostatin) expression was still positive
  • PDX-1 gene expression was negative
  • ⁇ -actin gene expression was negative
  • the stem cell marker Nestin gene expression was still positive. This indicates that in the model used in the present invention, although the islet ⁇ fine packets are severely damaged, nestin-positive pancreatic stem cells are still present in the pancreatic tissues of diabetic animals in the remaining limb islands (see Fig. 1A).
  • Example 2 Isolation of the remaining islet cell mass in the pancreatic tissue of the Tang dynasty animal
  • the pancreatic tissue obtained in Example 1 was cut into a lmm 3 tissue block, and the 3 mg/ml type V collagenase was digested at 37 ° C for 10 minutes. Then, add an equal volume of ice-cold Hank's solution to terminate the digestion, centrifuge at 1500 rpm for 2-3 minutes, remove the supernatant, wash the ice-cold Hank's solution for 3 times, filter with 100 mesh steel mesh, remove the large tissue, and separate the cells.
  • the pellet was inoculated in a 60 mm bacterial culture i containing medium A for 3 days, and was blown once a day with a 10 ml pipette to prevent the fine packets from adhering to the wall, and then the islet cell mass was picked under a dissecting microscope.
  • the isolated islet cell mass is a floating circular or elliptical islet cell mass in the bacterial culture Jni.
  • Multi-angled stellate cells (islet stem cells or pancreatic precursor cells) were subjected to fine-month immunohistochemistry.
  • the immunohistochemical staining of the fine JJ package was as follows: Culture the fine monthly bag at 4. C 4% paraformaldehyde was fixed for 20 minutes, then washed three times with 0.01 M PBS (culture cells were seeded on gelatin-coated coverslips), and 5% blocked with normal goat serum for 1 hour at room temperature, then 4. C-antibiotic incubation was overnight, washed three times with 0.01 MPBS, incubated with fluorescent secondary antibody for 2 hours at room temperature, washed three times with 0.01 MPBS, and then observed under Nikon2000U fluorescence.
  • the antibodies of the primary antibody and the secondary antibody were the same as in Example 1. Multi-angle stellate cells were subjected to RT-PCR amplification. The following methods were used for RT-PCR analysis: The fine-packed total RNA was extracted by TRIZOL method, 2 g of total RNA was reverse-transcribed into cDNA, and the RT-PCR reaction system was 50 mM Tris. -HCl (pH 8.3), 50 mM KCK 10 mM MgCl 2 , 10 mM DTT.
  • the PCR reaction system consisted of 10 mM Tris-HCK 50 mM KC1, 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 ⁇ ⁇ sense and antisense primers. , 6.25 U Taq DNA polymerase, and 3 ⁇ g cDNA.
  • the denaturation temperature is 94 °C for 45 seconds, and the annealing temperature is determined according to the specific primer.
  • the time is 30 seconds, and the extension temperature is 72 °C. minute.
  • the PCR product was separated by 1.5% gel electrophoresis.
  • Primer sequence Insulin upstream primer: TCA CAC CTG GTG GAA GCT C (SEQ ID NO: 19), downstream primer: ACA ATG CCA CGC TTC TGC (SEQ ID NO: 20) ( 179 bp )
  • Upstream Primer of Somatostatin GTT TCT GCA GAA GTC TGG G (Sequence ⁇ 'J 21 )
  • Downstream Primer AGT TCT TGC AGC CAG CTT TG (Order ⁇ ' J 22 ) ( 223 bp )
  • PDX-1 upstream primer GGA TGA AGT CTA CCA AAG CTC ACG C (Sequence ⁇ 'J 23 ), downstream primer: CCA GAT CTT GAT GTG TCT CTC GGT C (Serial ll 24 ) (218 bp).
  • Nestin upstream primers AGA GGG GAA TTC CTG GAG (sequence 25) and CTG AGG ACC AGG ACT CTC TA (sequence ⁇ 'J 26 ) (496 bp).
  • ABCG2 upstream primer GGT CTC AGG AAG ACT TAT GT (sequence 27), downstream primer: AAG GAG GTG GTG TAG CTG AT (preface ⁇ ' J 28 ) ( 323 bp ).
  • ⁇ primer CTT AAA TTG GAC TCC TAG AT (SEQ ID NO: 29), downstream primer: GGA TTT GGA ATG GCA TGC GG (sequence 30) (280 bp).
  • Glut2 upstream primer TCC TGG CCT TTA CCC TGT TTA C (Sequence ⁇ 'J 31 ), downstream primer: CAG ACG GTT CCC TTA TTG TTT C (Sequence ⁇ 'J 32 ) (209 bp ).
  • ⁇ -actin upstream primer TGGCACCACACCTTCTACAATGAGC (Sequence ⁇ 'J 33 ), downstream primer: GCACAGCTTCTCCTTAATGTCACGC (Sequence ⁇ 'J 34 ) (396bp).
  • the results of immunohistochemistry showed that these polygonal cells were nestin gene positive cells, as shown in Figure 3B.
  • the RT-PCR results are shown in the PPC on the left side of Figure 3A.
  • the following genes were positive: nestin, ABCG2, ⁇ -actin, and the following genes were negative: PDX-1, ISL-1 , Glut-2, insulin gene, growth hormone p-systemin gene (Somatostatin).
  • Example 4 Incubation of islet stem cells into brain gland endocrine cells
  • the star islet stem cells obtained in Example 3 were digested with 0.25% trypsin, washed in Hank's solution, and then seeded in medium containing medium.
  • a pre-islet-like structure pre-ICC
  • the medium was changed to medium D, but still in bacterial culture, cultured for about 4-6 days to obtain pancreatic endocrine cells of ICC structure.
  • the specific formulation we used was Formulation 7, but the other formulas mentioned in the text were also used to obtain the endocrine sac.
  • the culture in the culture dish containing the medium C may be 2-5 days, as shown in the figure, respectively, for 2 days of culture (Fig. 6A) and 5 days (Fig. 6B).
  • the resulting islet-like structure According to the experimental results, pancreatic endocrine cells can be obtained by culturing for 4-15 days in medium D (formulation 7), as shown in the figure, for 10 days (Fig. 6 C ) and 15 days (Fig. 6D ). The pancreas is secreted finely.
  • Medium C medium C
  • the ICC-structured squamous cell endocrine cells were subjected to RT-PCR amplification.
  • the following methods were used for RT-PCR analysis: Total RA of cells was extracted by TRIZOL method, and total RNA of 24 g was reverse-transcribed into cDNA.
  • the reaction system of RT-PCR consisted of 50 mM Tris-HCl ( ⁇ 8 ⁇ 3), 50 mM KCl, 10mM MgCl 2 , 10mM DTT, 0.5mM spermidine, 20U Rnasin, 0.5mM dNTP, 0.5 ⁇ g 01igo(dT) 15 , 50U SuperscriptTM II RT ( Invitrogen ), and nuclease-free water, then the reaction system is at 42 . C water bath for 60 minutes and 72 °C water bath for 15 minutes. The target gene was then amplified by PCR at 30-45 cycles.
  • the PCR reaction system consisted of 10 mM Tris-HCK 50 mM KCK 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 ⁇ M sense and antisense primers, 6.25 U Taq DNA polymerization drunk, and 3 ⁇ g cDNA.
  • the denaturation temperature was 94 ° C for 45 seconds
  • the annealing temperature was determined according to the specific primer
  • the time was 30 seconds
  • the extension temperature was 72 ° C for 7 minutes.
  • the PCR product was separated by 1.5% gel electrophoresis.
  • RT-PCR analysis showed that ICC compared with PPC, ICC nestin gene, ABCG2 gene expression decreased or disappeared, and islet related genes began to express, insulin, limb glucagon, somatostatin and PDX-1, ISL1, GLUT2 Isogenic expression was positive, see Figure 3A.
  • Immunohistochemistry showed that insulin-positive cells in ICCs were mainly distributed in the center of ICC and glucagon-positive cells were mainly distributed in the periphery of ICC, as shown in Figure 3C. Determination of insulin and C-peptide content Select 200 normal islets, Pre-ICC (before entering medium D) or ICC (refer to ICC after medium D differentiation), and place in a 0.5 ml small centrifuge tube.
  • FIG. 4 shows Pre-ICCs, ICCs and islets normal insulin (FIG. 4A) at high glucose 30mM and C-peptide (Fig. 4 B) of the secretion.
  • the secretion of insulin was ( ⁇ ⁇ / ml): pre-ICCs, 3.47 ⁇ 1.55; ICC, 45.02 ⁇ 2.94; Yueyi Island, 330.63 ⁇ 31.70.
  • RNA of cells or tissues was extracted by TRIZOL method, 2 g of total RNA was reverse-transcribed into cDNA, and RT-PCR reaction system consisted of 50 mM Tris-HCl (pH 8.3), 50 mM KCK 10 mM.
  • the PCR reaction system consisted of 10 mM Tris-HCK 50 mM KC1, 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 ⁇ ⁇ sense and antisense primers. (Sense and antisense primer), 6.25 U Taq DNA polymerase, and 3 ⁇ g cDNA.
  • the denaturation temperature was 94 °C for 45 seconds, and the annealing temperature was determined according to the specific primer, the time was 30 seconds, and the extension temperature was 72 minutes for 7 minutes.
  • the standard curve of PCR amplification of each gene (i.e., the relationship between the number of amplified DNA fragments and the number of cycles) was established by Real-time PCR (MJ Research, CFD-3220). In order to compare the transcription levels of individual genes, the number of cycles used in each PCR is The maximum number of cycles on the Real-time PGR standard curve.
  • the PCR product was separated by 1.5% gel electrophoresis.
  • Glucagon upstream primer ATTCACAGGGCACATTCACC (sequence 37), downstream primer: AACAATGGCGACCTCTTCTG (sequence 38) (260bp).
  • Somatostatin upstream primer GCTGCTGTCTGAACCCAAC (Sequence 391 39), downstream primer: CGTTCTCGGGGTGCCATAG (SEQ ID NO: 40) ( 138bp )
  • PDX-1 upstream primer TGATACTGGATTGGCGTTGT (SEQ ID NO: 41), downstream primer: GCATCAATTTCACGGGATCT (SEQ ID NO: 42) (270bp)
  • Nestin upstream primer AAGAGCTGGAGGGCGTGGTG (sequence 43) and TCCTGATAGCCGCGCACTG (sequence ⁇ 'J 44 ) (328bp)
  • ABCG2 upstream primer GGCCTCAGGAAGACTTATGT (sequence 45), downstream primer: AAGGAGGTGGTGTAGCTGAT (prologue 'J 46 ) ( 342bp )
  • Isll upstream primer TGTTTGAAATGTGCGGAGTG (prologue 'J 47 ), downstream primer: GTTCTTGCTGAAGCCGATG (prologue 'J 48 ) ( 144bp )
  • Glut2 on i primer TTGCTGGAAGAAGCATATCAGG (SEQ ID NO: 49)
  • downstream primer TGACTAATAAGAATGCCCGTGAC (SEQ ID NO: 50)
  • ⁇ -actin upstream primer TGGCACCACACCTTCTACAATGAGC (SEQ ID NO: 51)
  • downstream primer GCACAGCTTCTCCTTAATGTCACGC (SEQ ID NO: 52) (396bp).
  • Fig. 7 The results of RT-PCR analysis of pancreatic tissue of the animal model of the present invention are shown in Fig. 7, in which the left side is the result of pancreatic tissue cells of normal monkeys, and the right side is the result of pancreatic tissue of diabetic monkeys.
  • insulin gene Insulin
  • Glucagon glucagon gene
  • somatostatin gene did not change significantly
  • PDX-1 gene did not change significantly.
  • ⁇ -actin gene the expression of nestin gene was increased in stem cells, and the expression of Glut-2 gene was decreased.
  • Example 6 Detection of transcriptional levels of individual genes of ICCs after multi-angled stem cells and induced differentiation The detection of the transcriptional levels of individual genes of ICCs of the multi-angled stellate pancreatic stem cells of Example 3 and the induced differentiation of Example 4 was carried out.
  • the target gene is then amplified by PCR at 30-45 cycles, PCR
  • the reaction system consists of 10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100 1.75 mM MgCl 2 , 0.4 mM dNTP 0.2 ⁇ M sense and antisense primer, 6.25 U Taq DNA, and 3 ⁇ g cDNA.
  • the denaturation temperature was 94 °C for 45 seconds, and the annealing temperature was determined according to the specific primer, the time was 30 seconds, and the extension temperature was 72 °C for 7 minutes.
  • the standard curve of PCR amplification of each gene (i.e., the relationship between the number of amplified DNA fragments and the number of cycles) was established by Real-time PCR (MJ Research, CFD-3220). In order to compare the transcription levels of individual genes, the number of loops used for each PCR is the maximum number of loops on the Real-time PCR standard curve.
  • the PCR product was passed through 1.5%; the primer sequence was separated by electrophoresis: insulin upstream primer: GCAGCCTTTGTGAACCAACAC (sequence 53), downstream primer: CCCCGCACACTAGGTAGAGA (sequence 54) (67bp)
  • Somatostatin upstream primer GCTGCTGTCTGAACCCAAC (SEQ ID NO: 55), downstream primer: CGTTCTCGGGGTGCCATAG (Sequence ⁇ 'j 56 ) (138bp).
  • PDX-1 upstream primer TGATACTGGATTGGCGTTGT (SEQ ID NO: 57), downstream primer: GCATCAATTTCACGGGATCT (SEQ ID NO: 58) (270bp)
  • Nestin upstream primer A AGAGCTGGAGGGCGTGGTG (sequence
  • ABCG2 upstream primer GGCCTCAGGAAGACTTATGT (SEQ ID NO: 61), downstream primer: AAGGAGGTGGTGTAGCTGAT (Sequence ⁇ 'J 62 ) ( 342b )
  • Isll upstream primer TGTTTGAAATGTGCGGAGTG (prologue 'J 63 ), downstream primer: GTTCTTGCTGAAGCCGATG (sequence 64) : ( 144bp )
  • Glut2 upstream primer TTGCTGGAAGAAGCATATCAGG (sequence
  • ⁇ -actin upstream primer TGGCACCACACCTTCTACAATGAGC (SEQ ID NO: 67)
  • downstream primer GCACAGCTTCTCCTTAATGTCACGC (Sequence ⁇ 'J 68 ) (396bp).
  • FIG. 9 shows that insulin is stimulated by ICC and adult islets at different concentrations of glucose or with 1 OmM L-arginine ( Figures 9A1, 9A2)
  • the secretion of C-peptide was as follows: Under the stimulation of different concentrations of glucose, the amount of insulin secretion was (mIUx lO- 6 /min/ICC or islet): under glucose stimulation of OmM concentration: normal Insulin secretion of adult islets was 0.2174 ⁇ 0.0632, ICC was 0.0561 ⁇ 0.0258; under glucose stimulation of 5.6 mM: insulin secretion of adult islets was 0.9305 ⁇ 0.0896, ICC was 0.0845 ⁇ 0.0339; glucose stimulation at 16.7 mM the: amount of insulin secreted into body down islands is 2.0907 ⁇ 0.2644, ICC 0.1 XIE ⁇ 0.0542; secretion C- peptide is (pmolx l0- 6 / min / islet or ICC): at OmM concentrations of glucose stimulation: adult The
  • the amount of insulin secreted was (mlUx lO- 6 / Min/ ICC or islet):
  • the insulin secretion of adult islets was 0.6003 ⁇ 0 ⁇ 0419, ICC was 0.0641 ⁇ 0.0279; at the same time 5.6mM glucose and 10mML-arginine Under stimulation, the insulin secretion of adult islets was 2.7675 ⁇ 0.8867, ICC was 0.1809 ⁇ 0.0211.
  • the in vitro cultured pancreatic endocrine cells can be transplanted to diabetic animals in the same or allogeneic manner, so that diabetic animals can get rid of the dependence of insulin injection. It also solves the problem of rejection of allogeneic transplantation and insufficient source of thousands of cells.
  • the primer sequences mentioned in the present invention are artificially synthesized (which can be synthesized by Shanghai).

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Abstract

Methods of isolating and culturing pancreatic stem cells and differentiating the pancreatic stem cells into pancreatic endocrine cells that can secret insulin, comprises: isolating pancreas islet cell mass from the pancreas of animals which have diabetes, culturing the isolated cell mass in medium A for 2 to 5 days; culturing the isolated cell mass in medium B for 6 to 20 days to yield star pancreas islet stem cells; subculturing the star pancreas islet stem cells to yield pancreas islet stem cells. Such pancreas islet stem cells are inoculated onto medium B and are cultured for 4 to 15 days. Then the medium C is substituted for the medium D, and the pancreas islet stem cells are cultured for further 4 to 15 days to yield the pancreatic endocrine cells that can secret insulin.

Description

经培养的胰腺细胞及其  Cultured pancreatic cells and
培养方法和用途  Culture method and use
技术领域 本发明涉及干细胞， 尤其涉及从糖尿病动物 （特另 ij是 P甫乳 动物）胰腺組织分离得到的胰岛细胞团中培养和扩增胰岛干细 胞， 并将其进一步资导分化成为胰腺内分泌细胞的方法。 背景技术 从 1998年， James Thomson首次成功分离培养人胚胎干细 胞以来， 由于干细胞对将来疾病的治疗有着重要的应用前景， 因此干细胞技术毋庸质疑已成为当今生物科技领域的研究热点 之一。 而中国目前有 3000多万糖尿病患者， 而且正以每年 75 万新患者的速率递增， 糖尿病现已成为心脑血管病、 肿瘤之后 的 "人类第 3大杀手" 严重危及人类健康。 I型糖尿病（其也被 称作胰岛素依赖型糖尿病） 的患者必须每天依靠注射胰岛素来 维持。 虽然最近在加拿大 Edmonton, 研究人员采用新的免疫抑 制疗法使胰岛移植取得很好的疗效， 但该疗法仍然没有解决组 织供体来源不足的问题。 干细胞是一类能够自我复制、 自我更新并具有分化为相应 组织细胞潜能的细胞， 因此发现一种能够在体外大量扩增并能 分化为胰岛 β细胞的干细胞， 已成为目前的一个研究重点。 目前研究已证实，在体外可分化为胰岛 β细胞的干细胞主要 有以下三类： 胚胎干细胞 （ES )、 胰腺腺管来源的干细胞和胰 岛来源的干细胞。有关研究细节在 Shi,Y.， Hou, L.， Tang, F.， Jiang: W., Wang, P., Ding, M.， & Deng, H. ( 2005 ) . Inducing embryonic stem cells to differentiate into pancreatic β-cells by a novel three-step approach with activin A and all-trans retinoic acid. Stem Cells, 2005, 23, 656-662. ; Bonner-Weir, S.， Taneja, M.， Weir, G. C, Tatarkiewicz, ., Song, K. H., Sharma, A., & O'Neil, J. J.BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to stem cells, and more particularly to culturing and expanding islet stem cells from pancreatic islet cell populations isolated from pancreatic tissues of diabetic animals (specially ij is a P-milk animal), and further diluting them into pancreatic endocrine cells. method. BACKGROUND OF THE INVENTION Since the first successful isolation and cultivation of human embryonic stem cells by James Thomson in 1998, since stem cells have important application prospects for the treatment of future diseases, stem cell technology has undoubtedly become one of the research hotspots in the field of biotechnology. At present, there are more than 30 million diabetic patients in China, and they are increasing at a rate of 750,000 new patients per year. Diabetes has become a "human third killer" after cardiovascular and cerebrovascular diseases and cancer, which seriously endangers human health. Patients with type 1 diabetes (also known as insulin-dependent diabetes) must be maintained daily by injection of insulin. Although recently in Edmonton, Canada, researchers have used new immunosuppressive therapies to achieve good results in islet transplantation, the therapy still does not address the problem of insufficient tissue donor sources. Stem cells are a kind of cells that can self-replicate, self-renew and have the potential to differentiate into the corresponding tissue cells. Therefore, it has become a research focus to find a stem cell that can be expanded in vitro and can differentiate into islet β cells. At present, it has been confirmed that stem cells which can differentiate into islet β cells in vitro mainly have the following three types: embryonic stem cells (ES), pancreatic duct-derived stem cells, and islet-derived stem cells. Details of the research are in Shi, Y., Hou, L., Tang, F., Jiang : W., Wang, P., Ding, M., & Deng, H. ( 2005 ) . Inducing embryonic Stem cells to differentiate into pancreatic β-cells by a novel three-step approach with activin A and all-trans retinoic acid. Stem Cells, 2005, 23, 656-662. ; Bonner-Weir, S., Taneja, M., Weir, G. C, Tatarkiewicz, ., Song, KH, Sharma, A., &O'Neil, JJ
( 2000 ) . In vitro cultivation of human islets from expanded ductal tissue. Proceedings of the National Academy of Sciences of the United State of America, 97, 7999-8004.； Huang, H. X" & Tang, X. M. ( 2003 ) . Phenotypic determination and characterization of nestin- positive precursors derived from human fetal pancreas. Laboratory Investigation, 83, 539—547.; Wu, F.， Jagir, M., & Powell, J. S. ( 2004 ) . Long-term correction of hyperglycemia in diabetic mice after implantation of cultured human cells derived from fetal pancreas. Pancreas, 29, e23-e29.; Zulewski, H., Abraham, E. J., Gerlach, M. J., Daniel, P. B.， Moritz, W.， Muller, B.， Vallejo, M.， Thomas, M. Κ·, & Habener, J. F. ( 2001 ) · Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes, 50, 521 -533. , 等文章 中已有详尽描述。 胚胎干细胞是一种全能干细胞， 在适当的条件下它可分化 为体内任何组织的体细 JJ¾。 Lumelsky等人 4艮道， 通过体外五步 诱导的方法， 可将胚胎干细胞诱导分化为胰岛 β细胞， 而 Shi 等在他们的研究中 4艮道， 他们通过三步法可将胚胎千细胞诱导 成为胰岛 β细胞，并且这些细胞移植入糖尿病老鼠体内后可使动 物的体重增加， 存活时间延长， 血糖下降。 但是， 由于胚胎干 细胞存在目前尚无法解决的致瘤性问题， 因此目前离临床运用 还有一定的 3巨离。 对于胰腺腺管来源的干细胞， Bonner-Weir等在他们的研究 报告中报道， 在胰腺的腺管中存在一种 CK19 P曰性的干细胞， 此种细胞在有 KGF和 Matrigel存在的情况下，可大量扩增并分 化为胰岛 β细月包。 对于胰岛来源的干细胞， Zulewski等报道， 他们从正常胰 腺的胰岛团中分离培养出了一种表达神经干细胞标志 nestin蛋 白 （巢蛋白） 的细胞， 此种细胞可在体外大量扩增， 并且可在 exendin-4、 activin A、 HGF、 Betacellulin和 nicotiamide 的作用 下分化为胰月泉内分泌细包。 巢蛋白是一高分子量中间丝蛋白， 在分化细胞中巢蛋白表 达于纤维母细胞， 而未见表达于上皮细胞， 但在未分化始祖上 皮细胞中却可见其表达， 通过将表达有巢蛋白的胰腺导管未分 化上皮样细胞诱导分化成胰岛细胞的 1个亚群， 证明巢蛋白是 胰腺干细胞的 1种分子标记物， 它可能起着促进胰腺内分泌干 细包分化的作用。 参见 Peters K, Panienka R, Li J, Kloppel G, Wang R. Expression of stem cell markers and transcription factors during the remodeling of the rat pancreas after duct ligation. Virchows Arch 2005;446:56-63。 (2000) . In vitro cultivation of human islets from expanded ductal tissue. Proceedings of the National Academy of Sciences of the United State of America, 97, 7999-8004.; Huang, H. X"& Tang, XM ( 2003 ) . Phenotypic determination and characterization of nestin-positive precursors derived from human fetal pancreas. Laboratory Investigation, 83, 539-547.; Wu, F., Jagir, M., & Powell, JS (2004). Long-term correction of hyperglycemia in Pancreas, 29, e23-e29.; Zulewski, H., Abraham, EJ, Gerlach, MJ, Daniel, PB, Moritz, W., Muller, B., Vallejo , M., Thomas, M. Κ·, & Habener, JF ( 2001 ) · Multipotential nestin-positive stem cells isolated from adult pancreatic islets differentiate ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes. Diabetes, 50, 521 -533 , and other articles have been described in detail. Embryonic stem cells are a kind of pluripotent stem cells, which can differentiate under appropriate conditions. The body of any tissue in the body JJ3⁄4. Lumelsky et al. 4, through the in vitro five-step induction method, can induce embryonic stem cells to differentiate into islet β cells, and Shi et al. in their study 4, they passed three steps The method can induce embryonic cells into islet β cells, and transplanting these cells into diabetic mice can increase the body weight, prolong the survival time, and lower blood sugar. However, due to the tumorigenicity problems that are still unsolvable in embryonic stem cells, there are still a few major gaps away from clinical use. For pancreatic duct-derived stem cells, Bonner-Weir et al. reported in their study that there is a CK19 P-stem stem cell in the pancreatic duct. Such cells can be expanded and differentiated into islet β fine packets in the presence of KGF and Matrigel. For islet-derived stem cells, Zulewski et al. reported that they isolated a cell expressing the neural stem cell marker nestin (nestin) from the pancreatic islets of the normal pancreas, which can be expanded in vitro and can be The effects of exendin-4, activin A, HGF, Betacellulin and nicotiamide differentiate into pancreatic endocrine endocrine packets. Nestin is a high molecular weight intermediate filament protein. Nestin is expressed in fibroblasts in differentiated cells, but not in epithelial cells, but its expression is observed in undifferentiated ancestral epithelial cells by expressing nestin. Pancreatic duct undifferentiated epithelioid cells induce differentiation into a subset of islet cells, demonstrating that nestin is a molecular marker of pancreatic stem cells, which may play a role in promoting pancreatic endocrine dry fine-package differentiation. See Peters K, Panienka R, Li J, Kloppel G, Wang R. Expression of stem cell markers and transcription factors during the remodeling of the rat pancreas after duct ligation. Virchows Arch 2005;446: 56-63.
Huang & Tang, 2003 ; Zulewski et al., 2001文章中披露的 胰腺干细 J!包为胰岛来源的成体干细胞。 Zulewski等所用的组织 为正常的人或鼠的胰腺组织。 由于在实践中难以获得足够数量 的正常的人胰腺组织， 所以将该技术应用于临床时仍会面临来 源不足的问题。 因此， 需要解决胰岛移植中胰腺组织来源不足的问题， 还 需要解决异体移植中使用免疫抑制剂所带来的毒性问题。 T N2006/001669 发明内容 本发明的主要目的是用糖尿病动物自身残存的胰腺干细胞 在体外分离培养扩增后， 在诱导因子的作用下分化为有功能的 成熟的胰岛 β细胞， 然后再移植入该动物或者其它动物体内。 这 种自体移植的方法， 既解决了胰岛移植中胰腺组织来源不足的 问题，也解决了异体移植中使用免疫抑制剂所带来的毒性问题。 本领域技术人员可以理解， 利用根据本发明的方法获得的 可分泌胰岛素的成熟的胰岛 β细胞也可以进行异体移植 ,该异体 移植的方法可以解决胰岛移植中胰腺组织来源不足的问题。 根据本发明的第一方面， 提供了一种检测糖尿病动物胰腺 组织中是否存在胰腺干细胞的方法， 包括： a )免疫组化检测， 包括： 胰腺组织固定， 制作石蜡包埋切片， 封闭后接触一抗抗 体， 接触二抗抗体， 然后封片观察， 其中所述一抗抗体包括兔 抗巢蛋白抗体； b ) RT- PCR分析， 包括提取总 RNA, 逆转录 形成 cDNA, 然后利用引物进行 PCR扩增； c )根据步骤 a )和 b ) 的结果确定糖尿病动物胰腺组织中是否存在胰腺干细胞。 才艮据本发明的第二方面， 提供了一种从糖尿病动物胰泉组 织中分离残存的胰岛细胞团的方法， 包括以下步骤： a )将胰腺 組织剪碎成块； b )对块状的胰腺组织进行酶消化， 然后终止消 化； c ) 离心分离， 去上清， 清洗； d ) 得到分离后的胰岛细胞 团。 根据本发明的第三方面， 提供了一种胰岛干细胞的分离、 培养的方法， 包括以下步骤： a )将从糖尿病动物胰腺组织中分 离出的胰岛细胞团加入装有培养基 A的培养 中，培养 2-5天； b )将悬浮的胰岛细胞接种于含有培养基 B的培养亚中培养； c ) N2006/001669 经过 6-20天， 生长出星形胰岛干细胞； d ) 在所述星形胰岛干 细胞将汇合时进行传代， 得到胰岛干细胞。 所述培养基 A包括： ^出培养基、 緩冲液、 血清、 氨基酸 和抗生素等。 所述基础培养基包括 RPMI 1640等； 所述血清包 括 FBS等； 所述緩冲液包括 4-羟乙基哌嗪乙磺緩冲液、 丙酮酸 钠緩冲液等； 所述氨基酸包括 L-谷氨酸等； 所述抗生素包括青 霉素 -链霉素等。 培养基 A的一个实例为： RPMI 1640、 8-15% FBS、 10mM 4-羟乙基 ，秦乙横緩冲液、 ImM丙酮酸钠緩冲液、 2mM L-谷氨酸、 lOOu青霉素 -lOOug链霉素；另一实例为： RPMI 1640, FBS , HEPES ( 100X )， 丙酮酉吏 ] ( 100X ) , L-glutamine ( 100X ) , Antibiotic ( 100Χ )。 当然， 才艮据本发明披露的内容， 本领或的技术人员可以才艮据本领 i或的常识和具体情况对培养基 A的成分和数量做一些变动或修改， 例如， 基础培养基可以为 DMEM， 血清可以为马血清、 新生牛血清， 緩冲液可以为碳酸 氢钠緩冲液，所有这些变动和修改都应包括在本发明的范围内。 所述培养基 B包括： 基础培养基、 緩冲液、 血清、 氨基酸、 抗生素和生长因子。 所述基础培养基包括 RPMI 1640等； 所述 血清包括 FBS等； 所述缓冲液包括 4-羟乙基哌嗪乙横緩冲液、 丙酮酸钠緩冲液等； 所述氨基酸包括 L-谷氨酸等； 所述抗生素 包括青霉素—链霉素等； 所述生长因子包括碱性成纤维细胞生长 因子、 表皮细胞生长因子等。 培养基 B 的一个实例为： RPMI 1640、 8-15% FBS , 10mM 4-羟乙基哌"秦乙横緩冲液、 ImM丙 酮酸钠緩冲液、 2mM L-谷氨酸、 lOOu青霉素 -100ug链霉素、 10-20ng/ml碱性成纤维细胞生长因子、 10- 20ng/ml表皮细包生 长因子 ； 另一实例为： RPMI 1640 , FBS , HEPES ( 100X ) , 丙酮酉 钠， L-glutamine ( 100X ) ， Antibiotic ( 100X ) , Human bFGF , Human EGF。 当然， 根据本发明披露的内容， 本领域的 技术人员可以才艮据本领域的常识和具体情况对培养基 B的成分 和数量故一些变动或 ^ 改， 例如， 基础培养基可以为 DMEM, 血清可以为马血清、 新生牛 （胎牛） 血清， 緩冲液可以为碳酸 氢钠緩冲液，所有这些变动和修改都应包括在本发明的范围内。 根据本发明的第四方面， 提供了一种诱导胰岛干细胞 ( PPC ) 或者胰月泉前体细包分化为胰月泉内分泌细月包的方法。 联 合使用胰高血糖素样生长因子 （GLP-1 )、 肝细月包生长因子 ( HGF )、 betacellulin和烟酰胺等诱导分化因子， 诱导 PPC分 化为肢腺内分泌细胞。 一种将胰岛干细胞培养成为月夷腺内分泌 细胞的方法， 包括以下步驟： a )将胰岛干细胞接种于含有培养 基 C的培养皿中， 培养约 2-5天； b ) 将培养基 C更换为培养 基 D, 但仍然在培养 中， 培养大约 4-15天， 得到可分泌胰岛 素的胰月泉内分泌细月包。 其中所述培养亚为细菌培养 J ^或; '余有 0.01%多聚鸟氨酸或 多聚赖氨酸的细胞培养亚。 发明人发现， 在第四方面的方法中， 使用传统的细^；培养 "难以得到可分泌胰岛素的胰泉内分泌细 胞。 当使用细菌培养 i或涂有 0.01%多聚鸟氨酸或多聚赖氨酸 的细包培养 时可以获得好的效果。 所述培养基 C包括： 基础培养基、 培养添加剂、 白蛋白、 抗生素和生长因子。 其中所述基础培养基包括 DMEM 和 Nutrient Mixture Ham's F-12，； 所述培养添加剂包括 B27或者 (胰岛素 +转铁蛋白 +亚硒酸钠） ITS; 所述白蛋白包括 BSA; 所述抗生素包括青霉素-链霉素； 所述生长因子包括碱性成纤维 细包生长因子、 表皮细包生长因子。 所述培养基 C的一个实例 为： 48%DMEM、 48%Nutrient Mixture Ham's F-12\ 2%B27或 者 lg/L ITS (即 5mg/L胰岛素 +5mg/L转铁蛋白 +5ml/L亚硒酸 钠）、 0.05%-0.2% BSA、 lOOu青霉素 -lOOug链霉素、 10- 20ng/ml 碱性成纤维细胞生长因子、 10-20ng/ml表皮细胞生长因子； 另 一实例为： DMEM/F12 l:l(8mM glucose ) , B27( 50X, GIBCO), 0.075% BSA, bFGF 20ng/ml, EGF 20ng/ml, Antibiotics ( 100X, GIBCO)'。 当然， 本领域的技术人员可以才艮据本领域的常识和 具体情况对培养基 C的成分和数量做一些变动或修改， 所有这 些变动和 4爹改都应包括在本发明的范围内。 所述培养基 D包括： 基石出培养基、 培养添加剂、 白蛋白、 抗生素、 有 i秀导功能的多肽和生长因子。 所述基础培养基包括 DMEM和 Nutrient Mixture Ham's F-12，； 所述培养添力口剂包 括 B27或者 ITS (即胰岛素 +转铁蛋白 +亚硒酸钠 ) ； 所述白蛋 白包括 BSA; 所述抗生素包括青霉素-链霉素； 所述多肽和生长 因子包括： 烟酰胺、 GLP- HGF、 Betacellulin。 所述培养基 D 的一个实例为： 48%DMEM、 48%Nutrient Mixture Ham's F-12，、 2%B27 或者 lg/L ITS (即 5mg/L 胰岛素 +5mg/L 转铁蛋白 +5mg/L亚硒酸钠） .、 0.05%-0.1%BSA、 lOOu青霉素 -lOOug链 霉素、 lOmM 烟酰胺、 10-lOOnM GLP-1、 lOng/ml HGF、 500pmol/L Betacellulin; 另一实例为： DMEM/F12 1:1 ( 5.6mM glucose ), B27( 50X, GIBCO ), 0.075% BSA, lOmM nicotinamide, Antibiotics ( 100X, GIBCO ) , GLP-1 ( 7-36amide ) , HGF, Betacellulin。 当然， 本领域的技术人员可以才艮据本领域的常识 和具体情况对培养基 D的成分和数量做一些变动或 4爹改， 所有 这些变动和修改都应包括在本发明的范围内。 根据本发明的第五方面， 提供了一种利用前述的从糖尿病 动物胰腺组织中分离胰岛细胞团的方法得到的胰岛细胞团。 根据本发明的第六方面， 提供了一种利用前述的胰岛干细 胞的分离、 培养的方法得到的胰岛干细胞。 根据本发明的第^方面， 提供了一种利用前述的将胰岛干 N2006/001669 细胞培养成为胰腺内分泌细胞的方法得到的胰腺内分泌细胞。 附图说明 根据下文结合附图进行的描述， 本发明以及其另外的目的 和优点将更为明了， 其中： 图 1A显示正常和糖尿病动物胰月泉组织的 RT- PCR结果。 Huang & Tang, 2003; Zulewski et al., 2001 The dried pancreas J! package is an islet-derived adult stem cell. The tissue used by Zulewski et al. is normal human or murine pancreatic tissue. Since it is difficult to obtain a sufficient amount of normal human pancreatic tissue in practice, the application of this technique to clinical practice still faces problems of insufficient source. Therefore, it is necessary to solve the problem of insufficient source of pancreatic tissue in islet transplantation, and it is also necessary to solve the toxicity problem caused by the use of immunosuppressive agents in allogeneic transplantation. T N2006/001669 SUMMARY OF THE INVENTION The main object of the present invention is to use pancreatic stem cells remaining in a diabetic animal to be isolated and cultured in vitro, and then differentiate into functional mature islet β cells under the action of an inducing factor, and then transplanted into the pancreatic islet β cells. In animals or other animals. This method of autografting not only solves the problem of insufficient source of pancreatic tissue in islet transplantation, but also solves the toxicity problem caused by the use of immunosuppressants in allogeneic transplantation. It will be understood by those skilled in the art that the insulin-producing mature islet β cells obtained by the method according to the present invention can also be subjected to allogene transplantation, which can solve the problem of insufficient source of pancreatic tissue in islet transplantation. According to a first aspect of the present invention, there is provided a method for detecting the presence or absence of pancreatic stem cells in pancreatic tissue of a diabetic animal, comprising: a) immunohistochemical detection, comprising: pancreatic tissue fixation, preparation of paraffin embedded sections, and sealing after exposure Anti-antibody, contacted with secondary antibody, and then blocked, wherein the primary antibody comprises a rabbit anti-nestin antibody; b) RT-PCR analysis, including extraction of total RNA, reverse transcription to form cDNA, and then PCR amplification using primers c) determining the presence or absence of pancreatic stem cells in the pancreatic tissue of diabetic animals based on the results of steps a) and b). According to a second aspect of the present invention, there is provided a method for isolating residual islet cell mass from a diabetic animal pancreatic tissue comprising the steps of: a) cutting the pancreatic tissue into pieces; b) The pancreatic tissue is subjected to enzymatic digestion, and then the digestion is terminated; c) centrifugation, de-clearing, washing; d) obtaining isolated islet cell clusters. According to a third aspect of the present invention, there is provided a method for isolating and cultivating islet stem cells, comprising the steps of: a) adding islet cell clusters isolated from pancreatic tissue of a diabetic animal to a culture medium containing medium A, Incubate for 2-5 days; b) inoculate suspended islet cells in culture medium containing medium B; c) N2006/001669 Astrocytic stem cells are grown after 6-20 days; d) Pancreatic stem cells are obtained by passage when the star islet stem cells are confluent. The medium A includes: a medium, a buffer, a serum, an amino acid, an antibiotic, and the like. The basal medium includes RPMI 1640 or the like; the serum includes FBS or the like; the buffer includes 4-hydroxyethylpiperazine ethyl sulfonate buffer, sodium pyruvate buffer, etc.; and the amino acid includes L- Glutamate or the like; the antibiotic includes penicillin-streptomycin and the like. An example of medium A is: RPMI 1640, 8-15% FBS, 10 mM 4-hydroxyethyl, Qin E lateral buffer, 1 mM sodium pyruvate buffer, 2 mM L-glutamic acid, lOOu penicillin-lOOug Streptomycin; another example is: RPMI 1640, FBS, HEPES (100X), acetone oxime] (100X), L-glutamine (100X), Antibiotic (100Χ). Of course, according to the disclosure of the present invention, the skilled person or the skilled person can make some changes or modifications to the composition and quantity of the medium A according to the common sense and the specific conditions of the present, for example, the basic medium can be DMEM. The serum may be horse serum, newborn calf serum, and the buffer may be sodium bicarbonate buffer, and all such changes and modifications are included in the scope of the present invention. The medium B includes: a basal medium, a buffer, serum, an amino acid, an antibiotic, and a growth factor. The basal medium includes RPMI 1640 or the like; the serum includes FBS or the like; the buffer includes 4-hydroxyethyl piperazine B transverse buffer, sodium pyruvate buffer, etc.; the amino acid includes L-Valley The antibiotic includes penicillin-streptomycin or the like; the growth factor includes basic fibroblast growth factor, epidermal growth factor, and the like. An example of medium B is: RPMI 1640, 8-15% FBS, 10 mM 4-hydroxyethyl pipe "QIN Etra buffer, 1 mM sodium pyruvate buffer, 2 mM L-glutamate, lOOu penicillin - 100ug streptomycin, 10-20ng/ml basic fibroblast growth factor, 10- 20ng/ml epidermal growth factor; another example: RPMI 1640, FBS, HEPES (100X), sodium acetonide, L- Glutamine (100X), Antibiotic (100X), Human bFGF, Human EGF. Of course, according to the disclosure of the present invention, those skilled in the art can make the composition of the medium B according to the common knowledge and specific conditions in the art. And some changes or changes, for example, the basic medium can be DMEM, the serum can be horse serum, newborn cattle (fetal calf) serum, the buffer can be sodium bicarbonate buffer, all these changes and modifications It should be included in the scope of the invention. According to a fourth aspect of the present invention, there is provided a method of inducing differentiation of pancreatic islet stem cells (PPC) or pancreatic progenitor precursors into pancreatic spring endocrine cells. In combination with glucagon-like growth factor (GLP-1), hepatic serotonin growth factor (HGF), betacellulin, and nicotinamide to induce differentiation factors, PPC was induced to differentiate into limb gland endocrine cells. A method for culturing islet stem cells into endocrine cells of a melanogaster gland comprises the following steps: a) inoculation of islet stem cells in a culture dish containing medium C for about 2-5 days; b) replacing medium C with Medium D, but still in culture, cultured for about 4-15 days to obtain a secretory pericardium that can secrete insulin. Wherein the culture subculture is a bacterial culture J ^ or; a cell culture sub-containing 0.01% polyornithine or polylysine. The inventors have found that, in the method of the fourth aspect, it is difficult to obtain a pancreatic spring endocrine cell capable of secreting insulin when using the conventional method. When using bacterial culture i or coated with 0.01% polyornithine or poly-lyon A good effect can be obtained by fine-packing the amino acid. The medium C comprises: a basic medium, a culture additive, an albumin, an antibiotic, and a growth factor. The basal medium includes DMEM and Nutrient Mixture Ham's F-12. The culture additive includes B27 or (insulin + transferrin + sodium selenite) ITS; the albumin includes BSA; the antibiotic includes penicillin-streptomycin; and the growth factor includes basic fibril fine Growth factor, epidermal growth factor. An example of the medium C is: 48% DMEM, 48% Nutrient Mixture Ham's F-12\ 2% B27 or lg/L ITS (ie 5 mg/L insulin + 5 mg/ L transferrin + 5ml / L sodium selenite), 0.05% - 0.2% BSA, lOOu penicillin - lOOug streptomycin, 10- 20ng / ml basic fibroblast growth factor, 10-20ng / ml epidermal cell growth Factor; An example is: DMEM/F12 l: l (8 mM glucose), B27 (50X, GIBCO), 0.075% BSA, bFGF 20 ng/ml, EGF 20 ng/ml, Antibiotics (100X, GIBCO)'. Of course, those skilled in the art can make some changes or modifications to the composition and quantity of the medium C according to common knowledge and specific circumstances in the art, and all such variations and modifications should be included in the scope of the present invention. The medium D includes: a basal medium, a culture additive, albumin, an antibiotic, a polypeptide having an i-show function, and a growth factor. The basal medium comprises DMEM and Nutrient Mixture Ham's F-12, and the culture addition agent comprises B27 or ITS (ie, insulin + transferrin + sodium selenite); the albumin comprises BSA; Antibiotics include penicillin-streptomycin; the polypeptides and growth factors include: nicotinamide, GLP-HGF, Betacellulin. An example of such medium D is: 48% DMEM, 48% Nutrient Mixture Ham's F-12, 2% B27 or lg/L ITS (ie 5 mg/L insulin + 5 mg/L transferrin + 5 mg/L sub- Sodium selenate), 0.05%-0.1% BSA, lOOu penicillin-lOOug streptomycin, lOmM nicotinamide, 10-lOOnM GLP-1, lOng/ml HGF, 500 pmol/L Betacellulin; another example: DMEM/F12 1:1 (5.6 mM glucose), B27 (50X, GIBCO), 0.075% BSA, lOmM nicotinamide, Antibiotics (100X, GIBCO), GLP-1 (7-36amide), HGF, Betacellulin. Of course, those skilled in the art can make some changes or modifications to the composition and quantity of the medium D according to common knowledge and specific circumstances in the art, and all such changes and modifications are included in the scope of the present invention. According to a fifth aspect of the present invention, there is provided an islet cell mass obtained by the aforementioned method for isolating islet cell mass from pancreatic tissue of a diabetic animal. According to a sixth aspect of the present invention, there is provided an islet stem cell obtained by the method for isolating and culturing the islet stem cells described above. According to a second aspect of the present invention, there is provided a method of using the aforementioned tortoise N2006/001669 Cell culture of pancreatic endocrine cells obtained by the method of pancreatic endocrine cells. BRIEF DESCRIPTION OF THE DRAWINGS The invention and its additional objects and advantages will be more apparent from the following description taken in conjunction with the accompanying drawings in which: Figure 1A shows the results of RT-PCR of normal and diabetic animal pancreatic spring tissue.
图 1B显示正常猴胰腺组织中胰岛的免疫组化结果 （a, c, e ) 和糖尿病猴胰腺組织中胰岛的免疫組化结果（b , d, f )。 其中， a和 b分别表示正常猴和糖尿病猴的胰岛 β细包情况； c和 d分 别表示正常猴和糖尿病猴的胰高血糖素细胞（α细胞） 情况； e 和 f 分别表示正常猴和糖尿病猴的 δ细胞情况。 结果显示糖尿 病猴胰岛（3细胞几乎全部被破坏（b和 a ) , 胰高血糖素细胞明 显增多， 而分泌生长抑素的 δ细胞无明显改变。 图 1C 显示针 对 Nestin基因的免疫组化结果， a是正常猴的结果， b是糖尿病 猴的结果。 图 1C 显示， 对于正常猴和糖尿病猴， 胰腺组织中 均有 Nestin的表达， 只是 Nestin阳性细胞的分布有所变化， 在 正常猴的胰腺组织内， Nestin 阳性细胞分布于膝腺腺管和胰岛 团的周边， 而在糖尿病猴的胰腺组织中， Nestin P曰性细胞主要 分布于胰岛内， 并且数量明显增多。 图 2A、 2B、 2C、 2D显示胰岛、 胰腺前体细胞和胰岛样细 胞团的培养结果。 其中： 图 2A显示在 Petri dish (细菌培养亚） 中可见漂浮的圓形 或椭圓形胰岛细月包团； 图 2B 显示将得到的分离后的巢蛋白阳性的胰岛细胞团加 入培养基 A, 接种于 Petri dish中培养约 3天的结果； 001669 图 2C显示将图 2B中所示的胰岛细胞团接种于含有培养基 B的普通细胞培养 中培养 15天左右的结果； 图 2D显示将得到的星形胰岛干细胞（图 2( >斤示)用 0.25% 的肢酶消化下来后， Hank' s液中洗 1遍， 然后将细包接种于含 有培养基 C 的细菌培养 i中 3 天， 得到前类胰岛团样结构 ( pre-ICC：)。 图 3A为胰腺前侔细胞 ( PPC )和 ICC的 RT-PCR 结'果。 ' 图 3B显示胰腺前体细胞， 为巢蛋白阳性细包。 图 3C显示 ICC中胰岛素阳性细胞主要分布于 ICC的中央 而胰高血糖素阳性细胞主要分布于 ICC的周边。 图 4A和 4B显示 Pre-ICCs、 ICCs和正常夷岛团在 30mM 高糖刺激下月夷岛素和 C肽的分泌情况。 其中图 4A显示胰岛素 的分泌情况， 图 4B显示 C肽的分泌情况。 图 5A-5D显示胰腺组织细胞在 V型胶原酶中的消化和培养 后得到的胰岛细月包团的照片。 其中图 5A显示消化 5分钟， 培 养 2天的结果； 图 5B显示消化 5分钟， 培养 5天的结果； 图 5C显示消化 10分钟， 培养 2天的结果； 图 5D显示消化 10分 钟， 培养 5天的结果。 图 6A-6D显示才艮据实施例 4在含培养基 C和培养基 D的 培养 JUL中得到的类胰岛团样结构和胰腺内分泌细胞的照 '片。 图Figure 1B shows immunohistochemical results (a, c, e) of islets in normal monkey pancreatic tissue and immunohistochemical results (b, d, f) of islets in diabetic pancreatic tissue. Among them, a and b represent the islet β fine packets of normal monkeys and diabetic monkeys, respectively; c and d represent the glucagon cells (α cells) of normal monkeys and diabetic monkeys, respectively; e and f represent normal monkeys and diabetes, respectively. The delta cell condition of the monkey. The results showed that diabetic monkey islets (3 cells were almost completely destroyed (b and a), glucagon cells increased significantly, and δ cells secreting somatostatin did not change significantly. Figure 1C shows immunohistochemistry results for Nestin gene, a is the result of normal monkeys, and b is the result of diabetic monkeys. Figure 1C shows that for normal monkeys and diabetic monkeys, there is Nestin expression in pancreatic tissue, but the distribution of Nestin-positive cells varies, in pancreatic tissue of normal monkeys. Nestin-positive cells are distributed around the glandular gland and islet clusters, while in the pancreatic tissue of diabetic monkeys, Nestin P-sex cells are mainly distributed in islets and the number is significantly increased. Figure 2A, 2B, 2C, 2D The results of culture of islets, pancreatic precursor cells, and islet-like cell clusters are shown. Among them: Figure 2A shows floating circular or elliptical islet fines in a Petri dish; Figure 2B shows that The isolated nestin-positive islet cell mass was added to medium A, and inoculated in a Petri dish for about 3 days; 001669 Fig. 2C shows the results of inoculation of the islet cell mass shown in Fig. 2B in ordinary cell culture containing medium B for about 15 days; Fig. 2D shows the obtained star islet stem cells (Fig. 2 (> kg) After digesting with 0.25% of the limb enzyme, the Hank's solution was washed once, and then the fine packet was inoculated into the bacterial culture i containing the medium C for 3 days to obtain a pro-islet-like structure (pre-ICC:). Figure 3A shows the RT-PCR results of pre-pancreatic sputum cells (PPC) and ICC. ' Figure 3B shows pancreatic precursor cells, which are nestin-positive fine packets. Figure 3C shows that insulin-positive cells in ICC are mainly distributed in ICC. Central and glucagon-positive cells are mainly distributed around the periphery of ICC. Figures 4A and 4B show the secretion of prostaglandins and C-peptides by Pre-ICCs, ICCs, and normal islets stimulated by 30 mM high glucose. The secretion of insulin is shown, and Figure 4B shows the secretion of C-peptide. Figures 5A-5D show photographs of islet cell mass obtained after digestion and culture of pancreatic tissue cells in type V collagenase, wherein Figure 5A shows digestion 5 Minutes, 2 days of results; Figure 5B shows 5 minutes of digestion Figure 5C shows the results of digestion for 10 minutes and culture for 2 days; Figure 5D shows the results of digestion for 10 minutes and culture for 5 days. Figures 6A-6D show the use of medium C in accordance with Example 4 Culture medium D cultures the islet-like structure obtained in JUL and the photo of the pancreatic endocrine cells.
6A显示在培养基 C中培养 2天得到的类胰岛团样结构， 图 6B 显示在培养基 C中培养 5天得到的类胰岛团样结构。 图 6C显 示在培养基 D.中培养 10天得到的胰腺内分泌细胞，图 6D显示 在含培养基 D中培养 15天所得到的胰腺内分泌细胞。 图 Ί显示正常和糖尿病动物胰月泉组织的 RT- PCR结果。 图 8显示 PPC和 ICC的 RT-PCR结果。 图 9结果显示当 ICC 和成体胰岛在不同浓度的葡萄糖或在 同时含有 10mM L-精氨酸的刺激下， 胰岛素和 C-肽的分泌情 况， 其中图 9A1 显示 ICC在存在不同浓度的葡萄糖或葡萄糖 与 lOmM L-精氨酸同时存在的情况下胰岛素的分泌情况； 图 9A2 显示成体胰岛在存在不同浓度的葡萄糖或葡萄糖与 lOmM L-精氨酸同时存在的情况下胰岛素的分泌情况； 图 9B 1 显示 ICC在存在不同浓度的葡萄糖或葡萄糖与 lOmM 氨酸同时 存在的情况下 C-肽的分泌情况; 图 9B2 显示成体胰岛在存在 不同浓度的葡萄糖或葡萄糖与 lOmM L-精氨酸同时存在的情况 下 C-肽的分泌情况。 在以上图中， insulin表示胰岛素基因， Glucagon表示胰高 血糖素基因， Somatostatin表示生长抑素基因， PDX-1表示膝十 二指肠同源异型盒基因， nestin表示巢蛋白基因， β -actin表示 β肌动蛋白基因， " - " 、 "Normal" 表示正常猴， + STZ表 示糖尿病猴， Glut-2表示葡萄糖转运蛋白 -2基因， Pre- ICC表示 进入培养基 D之前的 ICC , ICC表示经培养基 D分化后的 ICC, Islet表示胰岛。 具体实施方式 对于患有糖尿病的动物（哺乳动物例如人或猴子）， 由于其 胰腺不能正常分泌胰岛素， 人们认为不能或难以从患有糖尿病 的动物的胰腺内分离出胰腺干细胞。 如果从异体动物的正常的 胰腺内分离出胰腺干细胞， 不可能进行自体移植， 排异反应的 问题依然存在， 并且从正常的胰腺内分离出的胰腺千细胞的来 N2006/001669 源问题也无法解决。 本发明的发明人发现在糖尿病动物的胰腺内仍然残存有巢 蛋白阳性的胰腺干细胞。 并且根据本发明提供的实施方式， 已 经成功地在体外分离和培养出这种细胞 , 该细胞具有以下干细 胞的分子生物学特征： 巢蛋白基因表达阳性、 ABCG2基因表 达阳性， 如图 1A-1 C所示。 在有丝分裂原存在的情况下 , 这些 细胞可长期扩增， 在特殊诱导分化因子的作用下可分化为胰岛 内分)必细胞。 6A shows an islet-like structure obtained by culturing in medium C for 2 days, and Fig. 6B shows an islet-like structure obtained by culturing in medium C for 5 days. Fig. 6C shows pancreatic endocrine cells obtained by culturing for 10 days in the medium D., and Fig. 6D shows pancreatic endocrine cells obtained by culturing for 15 days in the medium D. Figure Ί shows the RT-PCR results of normal and diabetic animal pancreatic spring tissues. Figure 8 shows the RT-PCR results of PPC and ICC. Figure 9 shows the secretion of insulin and C-peptide when ICC and adult islets are stimulated with different concentrations of glucose or with 10 mM L-arginine, wherein Figure 9A1 shows ICC in the presence of different concentrations of glucose or glucose. Insulin secretion in the presence of 10 mM L-arginine; Figure 9A2 shows insulin secretion in the presence of different concentrations of glucose or glucose and 10 mM L-arginine in adult islets; Figure 9B 1 Shows the secretion of C-peptide in the presence of ICC in the presence of different concentrations of glucose or glucose and 10 mM lysine; Figure 9B2 shows the presence of adult islets in the presence of different concentrations of glucose or glucose and 10 mM L-arginine The secretion of the lower C-peptide. In the above figure, insulin indicates the insulin gene, Glucagon indicates the glucagon gene, Somatostatin indicates the somatostatin gene, PDX-1 indicates the knee duodenal homeobox gene, nestin indicates the nestin gene, and β-actin indicates Β-actin gene, "-", "Normal" means normal monkey, + STZ means diabetic monkey, Glut-2 means glucose transporter-2 gene, Pre-ICC means ICC before entering medium D, ICC means cultured ICC after differentiation of base D, Islet represents islets. DETAILED DESCRIPTION OF THE INVENTION For animals with diabetes (mammals such as humans or monkeys), it is believed that it is not possible or difficult to isolate pancreatic stem cells from the pancreas of animals with diabetes because their pancreas does not normally secrete insulin. If pancreatic stem cells are isolated from the normal pancreas of the allogeneic animal, autologous transplantation is not possible, and the problem of rejection remains, and the pancreatic cells isolated from the normal pancreas come. N2006/001669 source issues cannot be resolved. The inventors of the present invention found that nestin-positive pancreatic stem cells still remain in the pancreas of diabetic animals. And according to the embodiments provided by the present invention, such cells have been successfully isolated and cultured in vitro, and the cells have the molecular biological characteristics of the following stem cells: positive expression of nestin gene and positive expression of ABCG2 gene, as shown in Fig. 1A-1C Shown. In the presence of mitogens, these cells can be expanded for a long period of time, and can differentiate into islet cells under the action of a specific differentiation-inducing factor.
ABCG2: ABCG2是 ABC超家族的一员， 它不但使肿瘤细 胞具有多种药物的耐药性， 而且最近发现它在维持千细胞的单 一特性方面 （即不分化方面）也有重要作用， 目前多数学者 4巴 它也看作干细胞的标志物之一。 根据本发明的方法得到的胰腺 干细胞中 ABCG2基因表达阳性。 ABCG2: ABCG2 is a member of the ABC superfamily. It not only makes tumor cells multi-drug resistant, but also recently found that it plays an important role in maintaining the unique characteristics of a thousand cells (ie, non-differentiation). 4 Barba is also considered as one of the markers of stem cells. The ABCG2 gene expression was positive in pancreatic stem cells obtained according to the method of the present invention.
CK19: 细包角质蛋白 19 ( cytokeratin- 19， CK-19 ) , Gmyr et β/发现成人 CK19阳性细胞能在体外再表达胰岛素促进因子 1 ( insulin promoter factor 1 )， 进一步表明人胰月泉多能前体细包 的存在， 也表明 CK- 19可能为胰腺前体细胞的分子标志之一。 CK19: fine keratin protein 19 (cytokeratin-19, CK-19), Gmyr et β/ found that adult CK19 positive cells can re-express insulin promoter factor 1 in vitro, further indicating that human pancreatic spring pluripotent The presence of a precursor packet also indicates that CK-19 may be one of the molecular markers of pancreatic precursor cells.
参见. Gmyr V, Kerr-Conte J, Belaich S, Vandewalle B, Leteurtre E, Vantyghem MC, Lecomte-Houcke M, Proye C, Lefebvre J, Pattou F. Adult human cytokeratin 19- positive cells reexpress insulin promoter factor 1 in vitro: further evidence for pluripotent pancreatic stem cells in humans. Diabetes 2000;49: 1671-1680。 See also. Gmyr V, Kerr-Conte J, Belaich S, Vandewalle B, Leteurtre E, Vantyghem MC, Lecomte-Houcke M, Proye C, Lefebvre J, Pattou F. Adult human cytokeratin 19- positive cells reexpress insulin promoter factor 1 in vitro : further evidence for pluripotent pancreatic stem cells in humans. Diabetes 2000; 49: 1671-1680.
但是， 根据本发明的方法得到的胰腺干细胞中 CK19基因表达 阴性。 pdx-1 是胰十二指肠同源异型盒基因 （pdx- 1 ) , 胰腺干细 胞发育过程中表达的第 1 种分子标记是 1 种同源区蛋白， 即 PDX-1。 pdx- 1对于出现在肠内胚层背侧及腹侧的胰腺萌芽的生 长分化起重要作用， pdx-1的纯合子缺失突变会导致胰腺无法形 成。 pdx-1阳性胚胎胰腺干细胞向胰腺内分泌细胞分化是多步骤 的， pdx-1阳性导管样细胞也可产生外分泌腺泡细胞。才艮据本发 明的方法得到的胰腺干细胞中 pdx-1基因表达阴性。 However, the CK19 gene expression was negative in pancreatic stem cells obtained according to the method of the present invention. Pdx-1 is a pancreatic duodenal homeobox gene (pdx-1). The first molecular marker expressed during pancreatic stem cell development is a homologous region protein. PDX-1. Pdx-1 plays an important role in the growth and differentiation of pancreas sprouting in the dorsal and ventral side of the intestinal endoderm. The homozygous deletion mutation of pdx-1 causes the pancreas to fail to form. Differentiation of pdx-1 positive embryonic pancreatic stem cells into pancreatic endocrine cells is multi-step, and pdx-1 positive duct-like cells can also produce exocrine acinar cells. The pdx-1 gene expression was negative in pancreatic stem cells obtained according to the method of the present invention.
ISL-1 : 是一个重要的转录因子,它在胰岛细胞的形成方面 起着重要的作用。 它所编码的蛋白能够结合到胰岛素基因的增 强区。 在成体的所有胰腺内分泌细胞均有表达。 才艮据本发明的 方法得到的胰腺干细胞中 ISL- 1基因表达阴性。 ISL-1: is an important transcription factor that plays an important role in the formation of islet cells. The protein it encodes binds to the potentiation region of the insulin gene. It is expressed in all pancreatic endocrine cells of the adult. The ISL-1 gene expression was negative in pancreatic stem cells obtained according to the method of the present invention.
Glut-2: 葡萄糖转运蛋白- 2, 是胰岛 β细胞的特定标记物， 它参与 β细胞的增殖和对葡萄糖的识别和转运。 才艮据本发明的 方法得到的胰月泉干细胞中 Glut-2基因表达阴性。 Somatostatin: 是生长抑素基因。 根据本发明， 将来源于糖尿病动物自身的干细胞进行体外 培养、分化， 可以将其用于糖尿病动物的自体干细包移植治疗， 也可以用于糖尿病的基因治疗中的载体细月包。 本发明的发明人发现， 虽然在糖尿病动物的胰岛内， β细胞 被完全破坏，但通过 3mg/ml V型胶原酶的消化，仍然可以分离 出残存的胰岛细胞团， 并且这些胰岛团贴壁后， 可迅速长成单 层扁平细胞， 这些细胞在 7- 8天时开始死亡， 但是同时一种多 角的星状细胞开始迅速增殖， 大概 14-16天这些细胞可汇合。 细胞免疫组 结果显示， 这些多角形细月包为巢蛋白阳性细月包， 但 CK19 染色为阴性， 故这种多角的星状细胞是胰岛干细胞 ( PPC ) 。 RT-PCR结果显示， 这些细胞除表达巢蛋白外， 还同 时表达另夕 1、一种干细胞的标志 ABCG2。 当这些细胞达到汇合 后， 继续生长， 可自发形成假胰岛团样结构 （ICC ) 。 当这些 细月包在分化培养基中， i秀导分化为 ICC, 免疫组化结果显示， 在 ICC结构中， 胰岛素阳性细胞分布于 ICC结构的中央， 而胰 高血糖素阳性细胞主要分布于 ICC结构的周边。 RT-PCR结果 显示， 与 PPC的 RT-PCR结果比较， 巢蛋白基因、 ABCG2基 因表达减弱或消失， 而胰岛相关基因开始表达， 胰岛素、 胰高 血糖素、生长抑素和 PDX-1、 ISL1、 GLUT2等基因表达呈阳性， 如图 3A-C所示。 本发明中所诱导分化得到的 ICC在 30mM的 葡萄糖刺激下可合成分泌胰岛素和 C肽， 分泌量约为正常胰岛 的 1/8左右， 如图 4A、 4B所示。 根据本发明的一种检测糖尿病动物胰腺组织中胰腺干细胞 的方法， 包括： a )免疫组化检测， 包括： 胰腺组织固定， 制作 石增包埋切片， †闭后接触一抗抗体， 接触二抗抗体， 然后封 片观察， 其中所述一抗抗体包括兔抗巢蛋白抗体； b ) RT- PCR 分析， 包括提取总 R A, 形成 cDNA， 然后利用引物进行 PCR 扩增； c )根据步驟 a ) 和 b ) 的结果确定糖尿病动物胰腺组织 中是否存在胰岛干细胞。 胰岛干细胞的主要标志是巢蛋白。 如 果糖尿病动物胰腺组织中是存在巢蛋白表达， 则存在胰岛干细 胞。 才艮据本发明的第二方面， 提供了一种从糖尿病动物胰腺组 织中分离残存的胰岛细胞团的方法， 包括以下步 : a )将胰腺 组织剪碎成块； b )对块状的胰腺组织进行酶消化， 然后终止消 化； c ) 离心分离， 去上清， 清洗； d )得到分离后的胰岛细胞 团。 根据本发明的第三方面， 提供了一种胰岛干细胞的分离、 培养的方法， 包括以下步骤： a )将从糖尿病动物胰腺组织中分 离出的胰岛细胞加入装有培养基 A的培养 i中，培养 2-5天； b ) 将悬浮的胰岛细胞接种于含有培养基 B的培养亚中培养； c )经 过 6-20天， 生长出星形胰岛干细胞； d )在所述星形胰岛干细 胞将汇合时传代， 直到长满传代后的培养亚， 得到胰岛干细胞。 其中， 培养基 A包括： RPMI 1640 厂家： Invitrogen公司， 力口利 畐尼亚州， 美国） 、 8-15% FBS ( Fetal Bovine Serum, 胎 牛血清）（厂家： Invitrogen公司）、 10mM HEPES Buffer SolutionGlut-2: Glucose transporter-2, a specific marker of islet beta cells, is involved in the proliferation of beta cells and the recognition and transport of glucose. The expression of Glut-2 gene was negative in pancreatic stem cells derived from the method of the present invention. Somatostatin: is a somatostatin gene. According to the present invention, stem cells derived from diabetic animals themselves can be cultured and differentiated in vitro, and can be used for autologous dry-package transplantation treatment of diabetic animals, and can also be used for carrier fine-moon packets in gene therapy of diabetes. The inventors of the present invention found that although β cells are completely destroyed in the islets of diabetic animals, residual islet cell clusters can still be isolated by digestion of 3 mg/ml type V collagenase, and these islet colonies are attached. It can rapidly grow into a single layer of flat cells, which begin to die in 7-8 days, but at the same time a multi-angled stellate cell begins to proliferate rapidly, and these cells can confluence in about 14-16 days. The results of the cell immunization group showed that these polygonal fine moon packs were nestin-positive fine-moon packets, but the CK19 staining was negative, so the multi-angled stellate cells were islet stem cells (PPC). RT-PCR results showed that these cells, in addition to the expression of nestin, also expressed the flag ABCG2 of a stem cell. When these cells reach confluence and continue to grow, they can spontaneously form a pseudo-islet-like structure (ICC). When these In the differentiation medium, the i-ray is differentiated into ICC. The results of immunohistochemistry show that in the ICC structure, insulin-positive cells are distributed in the center of the ICC structure, while glucagon-positive cells are mainly distributed in the ICC structure. Surroundings. The results of RT-PCR showed that compared with the RT-PCR results of PPC, the expression of nestin gene and ABCG2 gene was weakened or disappeared, and islet related genes began to express, insulin, glucagon, somatostatin and PDX-1, ISL1. Gene expression such as GLUT2 was positive, as shown in Figures 3A-C. The ICC induced by differentiation in the present invention can synthesize insulin and C peptide under the stimulation of 30 mM glucose, and the secretion amount is about 1/8 of that of normal islets, as shown in Figs. 4A and 4B. A method for detecting pancreatic stem cells in pancreatic tissue of a diabetic animal according to the present invention comprises: a) immunohistochemical detection, comprising: pancreatic tissue fixation, making a stone-enriched embedded slice, contacting the primary antibody after the sputum closure, contacting the secondary antibody The antibody is then blocked for observation, wherein the primary antibody comprises a rabbit anti-nestin antibody; b) RT-PCR analysis, including extraction of total RA, formation of cDNA, followed by PCR amplification using primers; c) according to step a) and The results of b) determine the presence of islet stem cells in the pancreatic tissue of diabetic animals. The main hallmark of islet stem cells is nestin. If nestin expression is present in the pancreatic tissue of a diabetic animal, islet stem cells are present. According to a second aspect of the present invention, there is provided a method for isolating residual islet cell mass from pancreatic tissue of a diabetic animal, comprising the steps of: a) cutting the pancreatic tissue into pieces; b) treating the massive pancreas The tissue is subjected to enzymatic digestion, and then the digestion is terminated; c) centrifugation, de-clearing, washing; d) obtaining isolated islet cell clusters. According to a third aspect of the present invention, a method for isolating and cultivating islet stem cells, comprising the steps of: a) adding islet cells isolated from pancreatic tissue of a diabetic animal to culture i containing medium A, Incubate for 2-5 days; b) inoculate suspended islet cells in culture medium containing medium B; c) After 6-20 days, the star-shaped islet stem cells are grown; d) the islet stem cells are passaged when the star-shaped islet stem cells are confluent, until the subcultured sub-culture cells are obtained, and the islet stem cells are obtained. Among them, medium A includes: RPMI 1640 Manufacturer: Invitrogen, Liquenia, USA), 8-15% FBS (Fetal Bovine Serum, fetal bovine serum) (manufacturer: Invitrogen), 10mM HEPES Buffer Solution
( 4-羟乙基派漆乙横緩冲液） （厂家： Invitrogen 公司 ） 、 ImMSodium pyruvate (丙酉同酸 3緩中液） （厂家： Invitrogen 公司） 、 2mM L-glutamine ( L-谷氣酸） （厂家： Invitrogen公 司） 、 lOOuPenicillin-lOOugStreptomycin (青霉素-链霉素） （厂 家： Invitrogen公司 ）。 所述培养基 B包括： RPMI 1640 (厂家： Invitrogen公司）、 8-15% FBS ( Fetal Bovine Serum, 月台牛血清）(4-Hydroxyethylpyrene Ethylene Buffer) (Manufacturer: Invitrogen), ImMSodium pyruvate (Producer: Invitrogen), 2mM L-glutamine (L-glutamic acid) (Manufacturer: Invitrogen), lOOuPenicillin-lOOugStreptomycin (penicillin-streptomycin) (Manufacturer: Invitrogen). The medium B includes: RPMI 1640 (manufacturer: Invitrogen), 8-15% FBS (Fetal Bovine Serum, platform beef serum)
(厂家： Invitrogen公司 ) 、 10mM HEPES Buffer Solution ( 4- 羟乙基 ϋ乙横緩冲液）（厂家： Invitrogen公司）、 ImMSodium pyruvate (丙酮酸納緩冲液） （厂家： Invitrogen公司） 、 2mM L-glutamine ( L-谷氛酸 ) ( 厂家： Invitrogen 公司 ) 、 1 OOuPenicillin- 1 OOugStreptomycin (青霉素-链霉素） （厂家： Invitrogen 公司 ) 、 10-20ng/ml Human Recombinant Basic Fibroblast Growth Factor ( bFGF ) (碱性成纤维细包生长因子）(Manufacturer: Invitrogen), 10mM HEPES Buffer Solution (Manufacturer: Invitrogen), ImMSodium pyruvate (Manufacturer: Invitrogen), 2mM L -glutamine (L-glutamic acid) (Manufacturer: Invitrogen), 1 OOuPenicillin- 1 OOugStreptomycin (manufacturer: Invitrogen), 10-20ng/ml Human Recombinant Basic Fibroblast Growth Factor ( bFGF ) ( Alkaline fibroblast growth factor
( R&D Systems 公司， 明尼苏达州， 美国）、 10-20ng/ml Human Recombinant Epidermal Growth Factor ( EGF ) (表皮细胞生长 因子） （ R&D Systems 公司 ) 。 根据本发明的第四方面，提供了一种诱导 PPC分化为胰腺 内分泌细胞的方法。联合使用胰高血糖素样生长因子（GLP-1 ). 肝细胞生长因子（ HGF ), betacellulin和烟酰胺等秀导分化因子， 诱导 PPC分化为胰腺内分泌细胞。 一种将胰岛干细胞培养成为 胰腺内分泌细胞的方法， 包括以下步骤： a )将胰岛干细胞接种 于含有培养基 C的培养 中， 培养 2-5天； b ) 将培养基 C更 换为培养基 D, 但仍然在培养亚中， 培养大约 4-15天， 得到可 分泌胰岛素的月夷腺内分泌细胞。 其中， 所述培养基 C包括： 48%DMEM (厂家： Invitrogen 公司， 货号 1 1966 ) 、 48%Nutrient Mixture Ham's F-12' (厂家： Invitrogen公司， 货号 11765 ) 、 2%B27 (厂家： Invitrogen公 司 ) 或者 1 g/L ( 5mg/L 胰岛素 +5mg/L transferring+mgyliter selenium )ITS (厂家： Sigma公司，密苏里州，美国）、 0·05%-0.2% BSA ( 牛血清白 蛋白 ） （ 厂 家： Sigma 公司 ） 、 1 OOuPenicillin- 1 OOugStreptomycin (青霉素-链霉素） （厂家： Invitrogen 公司 ) 、 10-20ng/ml Human Recombinant Basic Fibroblast Growth Factor ( bFGF ) , (碱性成纤维细包生长因子） ( R&D Systems 公司 ) 、 10-20ng/ml Human Recombinant Epidermal Growth Factor ( EGF )， (表皮细包生长因子 ) ( R&D Systems 公司）； 或者包括： DMEM/F12 1 : 1 ( 8mM glucose ) , B27( 50X, GIBCO ) , 0.075% BSA, bFGF 20ng/ml, EGF 20ng/ml, Antibiotics ( 100X, GIBCO ) 。 其中所述培养基 D 包括： 48%DMEM (厂家： Invitrogen 公司， 货号 11966 ) 、 48%Nutrient Mixture Ham's F-12' (尸家： Invitrogen公司， 货号 11765 ) 、 2%B27 (厂家： Invitrogen公 司 )或者 ( 5mg/L胰岛素 +5mg/L transferring+mgyliter selenium ) ITS (厂家： Sigma公司） 、 0.05%- 0.1% BSA (牛血清白蛋白）(R&D Systems, Inc., Minnesota, USA), 10-20 ng/ml Human Recombinant Epidermal Growth Factor (EGF) (R&D Systems). According to a fourth aspect of the invention, a method of inducing differentiation of PPC into pancreatic endocrine cells is provided. Combined use of glucagon-like growth factor (GLP-1). Hepatocyte growth factor (HGF), betacellulin, and nicotinamide induce differentiation of PPC into pancreatic endocrine cells. A method for culturing islet stem cells into pancreatic endocrine cells, comprising the steps of: a) inoculation of islet stem cells in culture medium containing medium C for 2-5 days; b) replacing medium C with medium D, But still in the cultivation of sub-culture, culture for about 4-15 days, get Insulin-secreting endocrine cells of the meridian gland. Wherein, the medium C comprises: 48% DMEM (manufacturer: Invitrogen, article number 1 1966), 48% Nutrient Mixture Ham's F-12' (manufacturer: Invitrogen, article number 11765), 2% B27 (manufacturer: Invitrogen) Or 1 g/L (5 mg/L insulin + 5 mg/L transfer+mgyliter selenium) ITS (manufacturer: Sigma, Missouri, USA), 0. 05%-0.2% BSA (bovine serum albumin) (Manufacturer: Sigma), 1 OOuPenicillin- 1 OOugStreptomycin (penicillin-streptomycin) (Manufacturer: Invitrogen), 10-20ng/ml Human Recombinant Basic Fibroblast Growth Factor ( bFGF ) , (Basic Fibroblast Growth Factor) ( R&D Systems, Inc., 10-20 ng/ml Human Recombinant Epidermal Growth Factor (EGF), (Epis Microscopic Growth Factor) (R&D Systems); or include: DMEM/F12 1 : 1 ( 8 mM glucose ) , B27 ( 50X , GIBCO ), 0.075% BSA, bFGF 20ng/ml, EGF 20ng/ml, Antibiotics (100X, GIBCO). The medium D includes: 48% DMEM (manufacturer: Invitrogen, article number 11966), 48% Nutrient Mixture Ham's F-12' (corporate: Invitrogen, article number 11765), 2% B27 (manufacturer: Invitrogen) Or (5mg/L insulin +5mg/L transfer+mgyliter selenium) ITS (vendor: Sigma), 0.05%- 0.1% BSA (bovine serum albumin)
(厂家： Sigma公司 ) 、 1 OOuPenicillin- 1 OOugStreptomycin (青 霉素-链霉素） （厂家： Invitrogen公司） 、 10mM烟酰胺（厂 家: sigma公司 ) 、 10-lOOnM GLP-l ( 7-36amide ) (胰高血糖素 样肽 1, 7-36氨基酸残基） （厂家: sigma公司） 、 10ng/ml HGF(Manufacturer: Sigma), 1 OOuPenicillin- 1 OOugStreptomycin (penicillin-streptomycin) (Manufacturer: Invitrogen), 10 mM nicotinamide (manufacturer: sigma), 10-lOOnM GLP-l (7-36amide) (pancreas Glucagon-like peptide 1, 7-36 amino acid residues) (Manufacturer: sigma), 10ng/ml HGF
(肝细胞生长因子） （厂家： R&D Systems公司） 、 500pmol/L BetacellulinC厂家： R&D Systems公司）或者包括 DMEM/F12 1： 1(Hepatocyte growth factor) (Manufacturer: R&D Systems, Inc., 500pmol/L BetacellulinC manufacturer: R&D Systems) or including DMEM/F12 1: 1
( 5.6mM glucose )， B27 ( 50X, Invitrogen公司 ) , 0.075% BSA, lOmM nicotinamide , Antibiotics ( 100X, Invitrogen公司 ) ， 10-lOOnM GLP-K 7-36 amide )(厂家: sigma公司）， lOng/ml HGF (厂家： R&D Systems公司） ， 500pmol/L Betacellulin (厂家： R&D Systems公司） 。 实施例 1 糖尿病动物模型胰腺组织中 nestin 阳性细月包的 检测 患有糖尿病的食蟹猴胰腺组织的获得 麻醉： Ketamine 10mg/kg Atropine 0.04mg/kg 安定 lmg/kg 腹部备皮 于剑突下腹正中切口， 切开腹膜进入腹腔， 分离大网膜及 肠管， 于胃大弯下缘可见略带粉红色的胰腺， 猴子的胰尾大多 游离， 于胰尾边缘先缝一针， 然后其周围合包缝合一块大约 0.5Xlcm大小的一块胰腺组织， 放入含有抗生素的冰冷 Hank' s液中并洗 2遍。 对胰腺干细包的检测 (5.6 mM glucose), B27 (50X, Invitrogen), 0.075% BSA, lOmM nicotinamide, Antibiotics (100X, Invitrogen), 10-lOOnM GLP-K 7-36 amide ) (manufacturer: sigma), lOng/ml HGF (manufacturer: R&D Systems), 500pmol/L Betacellulin (manufacturer: R&D Systems). Example 1 Diabetic animal model Detection of nestin-positive fine-moon pack in pancreatic tissue Obtaining anesthesia of pancreatic tissue of cynomolgus monkey with diabetes: Ketamine 10 mg/kg Atropine 0.04 mg/kg diazepam 1 mg/kg Abdominal skin preparation in the lower abdomen of the xiphoid Incision, incision of the peritoneum into the abdominal cavity, separation of the greater omentum and intestine, a slightly pink pancreas visible at the lower edge of the stomach, the monkey's pancreatic tail is mostly free, a needle is first sewn at the edge of the pancreas, and then wrapped around it A piece of pancreatic tissue approximately 0.5Xlcm in size was sutured, placed in ice-cold Hank's solution containing antibiotics and washed twice. Detection of dried pancreas
1 )免疫组^■方法： 胰腺组织在 4°C 4%的多聚甲酸中固定 24小时，然后 0.01M PBS洗三遍（胰腺组织石虫普包埋切片） ， 5%封闭用正常羊血清 室温下封闭 1 小时， 然后 4。C一抗孵浴过夜， 0.01MPBS洗三 遍， 荧光二抗室温孵浴 2 小时， 0.01MPBS 洗三遍， 封片后于 Nikon2000U 荧光显 4敖镜下观察。 所用到的抗血清有： 兔抗 nestin抗体（厂家： Chemicon公 司， 加利福尼亚州， 美国）、 豚鼠抗胰岛素抗体（厂家： Zymed Laboratories公司， 南力口利 畐尼亚州， 美国） 、 小鼠抗胰高血糖 素抗体 (厂家： Sigma公司）、 兔抗生长抑素抗体 (DAKO公司， 丹 麦）。 荧光二抗有： Texas red 羊抗兔 IgG, Texas red 羊抗小鼠 Ig G, Texas red 羊抗月豕鼠 IgG, Cy2 羊抗兔 IgG， 所有荧光抗 体都按 1 : 200稀释。 兔抗 nestin抗体:主要用来检测 Nestin蛋白。 Nestin蛋白是 一种中等纤维骨架蛋白， 在神经发育的研究中被确定为神经干 细胞的标志物， 目前多数学者认为 Nestin蛋白也可作为胰腺干 细月 的标志物。 兔抗胰岛素抗体： 主要用来检测胰岛 β细胞， 胰岛 β细胞是 胰腺内分泌部的主要功能细胞， 它是体内唯一可分泌胰岛素的 细胞。 小鼠抗胰高血糖素抗体： 主要用来检测肢岛内的 α细胞， α 细胞是胰岛内分泌胰高血糖素的细胞。 兔抗生长抑素抗体： 主要用来检测胰岛内的 δ细包， δ细 胞是胰岛内分泌生长抑素的细胞。 结果： 本发明动物模型免疫组化结果显示，胰岛的 β细胞被完全破 坏， 同时残存在胰岛内的 a细胞明显增生。 Nestin阳性细胞在 正常情况下存在于胰岛团的边缘和胰腺腺管， 而在实施例的模 型胰腺内， nestin 阳性细胞主要存在于胰岛团内， 腺管内分布 明显减少。 图 1B显示正常猴胰腺組织中胰岛的免疫组化结果 （a、 c、 e ) 和糖尿病猴胰腺组织中胰岛的免疫组化结果 （b、 d、 f ) 。 结果显示糖尿病猴的胰岛（3细胞全部被破坏（b和 a ) , 胰高血 糖素细胞明显增多 （d和 c ) , 而分泌生长抑素的 δ细胞无明显 改变 （f和 e ) 。 图 1C显示针对 Nestin基因的免疫组化结果， a是正常猴的 结果， b是糖尿病猴的结果。 图 1C显示， 对于正常猴和糖尿病 狼， 胰腺组织中均有 Nestin的表达， 只是 Nestin阳性细胞的分 布有所变 , 在正常猴的胰月泉组织内， Nestin 阳性细包分布于 胰腺腺管和胰岛团的周边， 而在糖尿病猴的胰腺组织中 , Nestin 阳性细胞主要分布于胰岛内， 并且数量明显增多。 1) Immunization group ^■ Method: Pancreatic tissue was fixed in 4% polyureic acid at 4 ° C for 24 hours, then washed three times with 0.01 M PBS (pancreatic tissue stone insect embedded section), 5% blocked with normal goat serum Block for 1 hour at room temperature, then 4. C-antibiotic incubation was overnight, washed three times with 0.01 MPBS, incubated with fluorescent secondary antibody for 2 hours at room temperature, washed three times with 0.01 MPBS, and then observed under Nikon 2000U fluorescence. Antisera used were: rabbit anti-nestin antibody (manufacturer: Chemicon, California, USA), guinea pig anti-insulin antibody (manufacturer: Zymed Laboratories Inc., South Lili, USA), mouse anti-glucagon antibody (manufacturer: Sigma), rabbit anti-somatostatin antibody (DAKO, Denmark). The fluorescent secondary antibodies were: Texas red goat anti-rabbit IgG, Texas red goat anti-mouse Ig G, Texas red goat anti-moon mole IgG, Cy2 goat anti-rabbit IgG, all fluorescent antibodies were diluted 1:200. Rabbit anti-nestin antibody: mainly used to detect Nestin protein. Nestin protein is a medium fiber skeleton protein, which has been identified as a marker of neural stem cells in the study of neurodevelopment. At present, most scholars believe that Nestin protein can also be used as a marker for pancreatic stem. Rabbit anti-insulin antibody: It is mainly used to detect islet β cells. Pancreatic islet β cells are the main functional cells in the endocrine part of the pancreas. It is the only cell that can secrete insulin in the body. Mouse anti-glucagon antibody: It is mainly used to detect α cells in limb islands, and α cells are cells of islet endocrine glucagon. Rabbit anti-somatostatin antibody: mainly used to detect δ fine packets in islets, δ cells are cells of endocrine somatostatin in islets. Results: The immunohistochemical results of the animal model of the present invention showed that the β cells of the islets were completely destroyed, and the a cells remaining in the islets were obviously proliferated. Nestin-positive cells are normally present at the edge of the islet group and the pancreatic gland. In the model pancreas of the example, nestin-positive cells are mainly present in the islet group, and the distribution in the gland is significantly reduced. Figure 1B shows immunohistochemical results ( a , c , e) of islets in normal monkey pancreatic tissue and immunohistochemical results (b, d, f) of islets in diabetic pancreatic tissue. The results showed that the islets of diabetic monkeys (all 3 cells were destroyed (b and a), glucagon cells were significantly increased (d and c), and δ cells secreting somatostatin did not change significantly (f and e). The results of immunohistochemistry for the Nestin gene are shown, a is the result of normal monkeys, and b is the result of diabetic monkeys. Figure 1C shows that for normal monkeys and diabetic wolves, the expression of Nestin is found in pancreatic tissue, only the distribution of Nestin-positive cells. In the normal monkey's pancreatic spring tissue, Nestin-positive fine packets are distributed around the pancreatic duct and pancreatic islets. In the pancreatic tissue of diabetic monkeys, Nestin-positive cells are mainly distributed in islets, and the number is obvious. increase.
2 ) RT-PCR分析方法： 细月包或组织的总 RNA采用 TRIZOL法提取， 2 克的总 RNA被逆转录为 cDNA, RT- PCR的反应体系由 50 mM Tris-HCl (pH 8.3), 50 mM KC1, 10 mM MgCl2, 10 mM DTT, 0.5 mM spermidine, 20 U of Riiasin, 0.5mM dNTP, 0.5 μ g of 01igo(dT)15, 50 U of Superscript™ II RT(Invitrogen), 和无核酶水构成， 然后 反应体系在 42°C水浴 60分钟和 72°C水浴 15分钟。 然后通过 30-45循环的 PCR扩增目标基因， PCR的反应体系由 10 mM Tris-HCl, 50 mM KC1, 0.1%Triton X-100, 1.75 mM MgCl2, 0.4 mM dNTP, 0.2 μ Μ of sense and antisense primer, 6.25 U Taq DNA polymer¾se and 3 g of cDNA构成。 变' 1"生温度为 94°C 时 间为 45秒， 退火温度根据具体引物决定， 时间为 30秒， 延伸 温度为 72°C 时间为 7分钟。 PCR产物通过 1.5%的凝胶电泳分 离。 引物序列： 2) RT-PCR analysis method: Total RNA of fine moon pack or tissue is extracted by TRIZOL method, 2 g of total RNA is reverse-transcribed into cDNA, and RT-PCR reaction system consists of 50 mM Tris-HCl (pH 8.3), 50 mM KC1, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM spermidine, 20 U of Riiasin, 0.5 mM dNTP, 0.5 μg of 01igo(dT) 15 , 50 U of SuperscriptTM II RT (Invitrogen), and ribozyme The water was composed, and then the reaction system was subjected to a water bath at 42 ° C for 60 minutes and a water bath at 72 ° C for 15 minutes. The target gene was then amplified by PCR at 30-45 cycles. The PCR reaction system consisted of 10 mM Tris-HCl, 50 mM KC1, 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 μ Μ of sense and Antisense primer, 6.25 U Taq DNA polymer 3⁄4se and 3 g of cDNA. The temperature of '1' was 94 ° C for 45 seconds, the annealing temperature was determined according to the specific primer, the time was 30 seconds, and the extension temperature was 72 ° C for 7 minutes. The PCR product was separated by 1.5% gel electrophoresis. sequence:
Insulin上游引物： TCA CAC CTG GTG GAA GCT C (序列 1 ) , 下游引物： ACA ATG CCA CGC TTC TGC (序列 2 ) (179bp)。 Insulin upstream primer: TCA CAC CTG GTG GAA GCT C (SEQ ID NO: 1), downstream primer: ACA ATG CCA CGC TTC TGC (sequence 2) (179 bp).
Glucagon上游引物： ATT CAC AGG GCA CAT TCA CC (序列 3 ) ,下游引物： AAC AAT GGC GAC CTC TTC TG (序 列 4 ) (260bp)o Glucagon upstream primer: ATT CAC AGG GCA CAT TCA CC (SEQ ID NO: 3), downstream primer: AAC AAT GGC GAC CTC TTC TG (sequence 4) (260 bp) o
Somatostatin上游引物： GTT TCT GCA GAA GTC TGG G (序歹 'J 5 ) , 下游引物： AGT TCT TGC AGC CAG CTT TG (序 列 6 ) (223bp)。 Somatostatin upstream primer: GTT TCT GCA GAA GTC TGG G (Sequence 歹 'J 5 ), downstream primer: AGT TCT TGC AGC CAG CTT TG (Sequence 6) (223 bp).
PDX-1上游引物： GGA TGA AGT CTA CCA AAG CTC ACG C (序歹 'J 7 ) , 下游引物： CCA GAT CTT GAT GTG TCT CTC GGT C (序列 8 ) (218bp)。 PDX-1 upstream primer: GGA TGA AGT CTA CCA AAG CTC ACG C (Sequence 歹 'J 7 ), downstream primer: CCA GAT CTT GAT GTG TCT CTC GGT C (SEQ ID 8 ) (218 bp).
Nestin上游引物： AGA GGG GAA TTC CTG GAG (序列 9 )和 CTG AGG ACC AGG ACT CTC TA (序歹 'J 10 ) (496bp)。 Nestin upstream primers: AGA GGG GAA TTC CTG GAG (sequence 9) and CTG AGG ACC AGG ACT CTC TA (sequence 歹 'J 10 ) (496 bp).
ABCG2上游引物： GGT CTC AGG AAG ACT TAT GT (序 歹 ll 11 ) , 下游引物： AAG GAG GTG GTG TAG CTG AT (序 列 12 ) (323bp)。 ABCG2 upstream primer: GGT CTC AGG AAG ACT TAT GT (Sequence ll 11 ), downstream primer: AAG GAG GTG GTG TAG CTG AT (Sequence 12) (323 bp).
Isll上游引物: CTT AAA TTG GAC TCC TAG AT (序列 13 ) , 下游引物： GGA TTT GGA ATG GCA TGC GG (序列 14 ) (280bp)。 Isll upstream primer: CTT AAA TTG GAC TCC TAG AT (SEQ ID NO: 13), downstream primer: GGA TTT GGA ATG GCA TGC GG (SEQ ID NO: 14) (280 bp).
列 15 ) , 下游引物： CAG ACG GTT CCC TTA TTG TTT C (序 列 16 ) (209bp) β - actin上游引物： TGG CAC CAC ACC TTC TAC AAT GAG C (序列 17 ) , 下游引物： GCA CAG CTT CTC CTT AAT GTC ACG C (序歹 'J 18 ) (396bp)0 结果: 本发明动物模型胰腺组织的 RT-PCR 分析结果显示如图 1A所示， 其中左侧的是正常猴的胰腺组织细胞的结果， 右侧是 糖尿病动物的胰月泉组织的结果。 如图 1A 所示， 胰岛素基因 ( Insulin ) 表达转为阴性， 胰高血糖素基因 （ Glucagon ) 和生 长抑素基因 （Somatostatin )表达仍呈现阳性， PDX- 1基因表达 转为阴性， β -actin基因表达仍呈现阳性， 干细胞标志 Nestin 基因表达仍呈现阳性。 这说明在本发明使用的模型中， 虽然胰 岛 β细包被严重破坏， 但在残存的肢岛内， nestin阳性的胰腺干 细胞仍然存在于糖尿病动物的胰腺组织中 （见图 1 A ) 。 Column 15), downstream primer: CAG ACG GTT CCC TTA TTG TTT C (SEQ ID NO: 16) (209 bp) β-actin upstream primer: TGG CAC CAC ACC TTC TAC AAT GAG C (SEQ ID NO: 17), downstream primer: GCA CAG CTT CTC CTT AAT GTC ACG C (Sequence 歹 'J 18 ) (396 bp) 0 Results: The results of RT-PCR analysis of pancreatic tissue of the animal model of the present invention are shown in Fig. 1A, wherein the left side is the result of pancreatic tissue cells of normal monkeys, right The side is the result of the pancreatic spring tissue of diabetic animals. As shown in Figure 1A, insulin gene (Insulin) expression was negative, glucagon gene (Glucagon) and somatostatin gene (Somatostatin) expression was still positive, PDX-1 gene expression was negative, β-actin gene The expression was still positive, and the stem cell marker Nestin gene expression was still positive. This indicates that in the model used in the present invention, although the islet β fine packets are severely damaged, nestin-positive pancreatic stem cells are still present in the pancreatic tissues of diabetic animals in the remaining limb islands (see Fig. 1A).
实施例 2 唐尿病动物胰腺组织中分离残存的胰岛细胞团 将实施例 1中获得的胰腺组织剪碎成 lmm3大小的组织块， 3mg/ml的 V型胶原酶中 37°C消化 10分钟， 然后加入等体积的 冰冷的 Hank's液终止消化， lOOOrpm离心 2-3分钟后， 去上清， 冰冷 Hank's液中洗 3遍， 100目钢网过滤， 除去大块组织后， 将分离得到的细胞团接种于含有培养基 A的 60mm的细菌培养 i中 3天， 每天用 10ml的移液管吹打一次， 防止细包团贴壁， 然后于解剖镜下挑取胰岛细胞团。 参见图 2A, 分离得到的胰岛 细胞团在细菌培养 Jni中为漂浮的圆形或椭圆形胰岛细胞团。 另夕卜，根据实验数据，当在 V型胶原酶中的消 时间为 5-10 分钟， 并且细包团在培养亚中培养 2-5 天时， 均可以得到基本 相同的胰岛细胞团，以下分别为消化 5分钟，培养 2天 (见图 5A); 消化 5分钟， 培养 5天 (见图 5B); 消化 10分钟， 培养 2天（见 图 5C )； 消化 10分钟， 培养 5天得到的胰岛细胞团（见图 5D ) 实施例 3 腹岛干细胞的分离、 培养 将实施例 2中得到的分离后的巢蛋白阳性的胰岛细胞团再 加入培养基 A,接种于 60mm的 Petri dish中 3天，每天用 10ml 的移液管吹打细包一次， 防止胰岛贴壁。 3 天后' 将挑取的胰 岛细胞团接种于含有培养基 B的普通细胞培养 中培养 15天 左右，当生长出来的星形胰岛干细胞将汇合时传代，参见图 2B、 2C。 以下为培养基 A和培养基 B的成分， 在实施例 3中' 采用 的具体配方是配方 4, 但是采用文中提到的其他配方也可以同 样得到胰岛干细胞。 培养基 A Example 2 Isolation of the remaining islet cell mass in the pancreatic tissue of the Tang dynasty animal The pancreatic tissue obtained in Example 1 was cut into a lmm 3 tissue block, and the 3 mg/ml type V collagenase was digested at 37 ° C for 10 minutes. Then, add an equal volume of ice-cold Hank's solution to terminate the digestion, centrifuge at 1500 rpm for 2-3 minutes, remove the supernatant, wash the ice-cold Hank's solution for 3 times, filter with 100 mesh steel mesh, remove the large tissue, and separate the cells. The pellet was inoculated in a 60 mm bacterial culture i containing medium A for 3 days, and was blown once a day with a 10 ml pipette to prevent the fine packets from adhering to the wall, and then the islet cell mass was picked under a dissecting microscope. Referring to Figure 2A, the isolated islet cell mass is a floating circular or elliptical islet cell mass in the bacterial culture Jni. In addition, according to the experimental data, when the elimination time in the V-type collagenase is 5-10 minutes, and the fine-packed group is cultured in the culture sub-culture for 2-5 days, substantially the same islet cell cluster can be obtained, the following respectively For digestion for 5 minutes, culture for 2 days (see Figure 5A); digestion for 5 minutes, culture for 5 days (see Figure 5B); digestion for 10 minutes, culture for 2 days (see Figure 5C); digestion for 10 minutes, culture for 5 days to obtain islets Cell cluster (see Figure 5D) Example 3 Isolation and culture of the islet-derived stem cells The isolated nestin-positive islet cell mass obtained in Example 2 was further added to the medium A, and inoculated into a 60 mm Petri dish for 3 days, using a 10 ml pipette per day. Blow the fine bag once to prevent the islets from sticking. After 3 days, the picked islet cell pellet was inoculated in a normal cell culture medium containing medium B for about 15 days, and when the grown star islet stem cells were confluent, they were passaged, see Figures 2B and 2C. The following are the components of Medium A and Medium B. In Example 3, the specific formulation used was Formulation 4, but islet stem cells were also obtained using the other formulations mentioned herein. Medium A
成分 比例 厂家 Composition ratio manufacturer
RPMI 1640 86% InvitrogenRPMI 1640 86% Invitrogen
FBS 10% InvitrogenFBS 10% Invitrogen
HEPES ( 1M ) 1% Invitrogen 配方 1 HEPES ( 1M ) 1% Invitrogen Formula 1
丙酮酸 ] ( lOOmM ) 1% Invitrogen Pyruvate ] ( lOOmM ) 1% Invitrogen
L-谷氨酰胺 ( 200raM ) 1% Invitrogen lOOOOu青霉素 -lOOOOug链霉素 1% InvitrogenL-Glutamine (200raM) 1% Invitrogen lOOOOu Penicillin - lOOOOug Streptomycin 1% Invitrogen
RPMI 1640 81% InvitrogenRPMI 1640 81% Invitrogen
FBS 15% InvitrogenFBS 15% Invitrogen
HEPES ( 1M ) 1% Invitrogen 配方 2 HEPES ( 1M ) 1% Invitrogen Formula 2
丙酮酸 ] ( lOOmM ) 1% Invitrogen Pyruvate ] ( lOOmM ) 1% Invitrogen
L-谷氨醜胺 ( 200mM ) 1% Invitrogen lOOOOu青霉素 -lOOOOug链霉素 1% InvitrogenL-Glutamine Oxide ( 200 mM ) 1% Invitrogen lOOOOu Penicillin - lOOOOug Streptomycin 1% Invitrogen
RPMI 1640 88% InvitrogenRPMI 1640 88% Invitrogen
FBS 8% InvitrogenFBS 8% Invitrogen
HEPES ( 1M ) 1% Invitrogen 配方 3 HEPES ( 1M ) 1% Invitrogen Formula 3
丙酮酉 钠 ( lOOmM ) 1% Invitrogen Sodium acetonide ( lOOmM ) 1% Invitrogen
L-谷氨酰胺 （200mM ) 1% Invitrogen lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen 配 方 RPMI 1640 43ml Invitrogen 4(等同 FBS 5ml Invitrogen 于配方 HEPES ( 100X ) 0.5ml Invitrogen 1) L-Glutamine (200 mM) 1% Invitrogen lOOOOu Penicillin-lOOOOug Streptomycin 1% Invitrogen Formulation RPMI 1640 43ml Invitrogen 4 (equivalent to FBS 5ml Invitrogen in formula HEPES ( 100X ) 0.5ml Invitrogen 1)
丙酮酸钠 ( 100X ) 0.5ml Invitrogen Sodium pyruvate ( 100X ) 0.5ml Invitrogen
L-glutamine ( 100X ) 0.5ml InvitrogenL-glutamine ( 100X ) 0.5ml Invitrogen
Antibiotic ( 100X ) 0.5ml Invitrogen 基 B Antibiotic ( 100X ) 0.5ml Invitrogen Base B
成分 比例 厂家 Composition ratio manufacturer
RPMI 1640 86% InvitrogenRPMI 1640 86% Invitrogen
FBS 10% InvitrogenFBS 10% Invitrogen
HEPES ( 1M ) 1% Invitrogen 丙酉同酉 4内 ( lOOmM ) 1% Invitrogen 配方 1 L-谷氣酰胺 （200mM ) 1% Invitrogen lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen HEPES ( 1M ) 1% Invitrogen 酉 酉 酉 4 ( lOOmM ) 1% Invitrogen Formula 1 L-Glutamine (200mM ) 1% Invitrogen lOOOOu Penicillin - lOOOOug Streptomycin 1% Invitrogen
20ng/m R&D bFGF 1 Systems  20ng/m R&D bFGF 1 Systems
20ng/m R&D 20ng/m R&D
EGF 1 SystemsEGF 1 Systems
RPMI 1640 81% InvitrogenRPMI 1640 81% Invitrogen
FBS 15% InvitrogenFBS 15% Invitrogen
HEPES ( 1M ) 1% Invitrogen 丙酮酸钠 ( lOOmM ) 1% Invitrogen 配方 2 L-谷氣酰胺 ( 200mM ) 1% Invitrogen lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen HEPES ( 1M ) 1% Invitrogen Sodium Pyruvate ( lOOmM ) 1% Invitrogen Formulation 2 L-Glutamine ( 200 mM ) 1% Invitrogen lOOOOu Penicillin - lOOOOug Streptomycin 1% Invitrogen
20ng/m R&D bFGF 1 Systems  20ng/m R&D bFGF 1 Systems
20ng/m R&D 20ng/m R&D
EGF 1 SystemsEGF 1 Systems
RPMI 1640 88% InvitrogenRPMI 1640 88% Invitrogen
FBS 8% InvitrogenFBS 8% Invitrogen
HEPES ( 1M ) 1% Invitrogen 丙酮酸钠 ( lOOmM ) 1% InvitrogenHEPES ( 1M ) 1% Invitrogen Sodium Pyruvate ( lOOmM ) 1% Invitrogen
L-谷氨酰胺 ( 200mM ) 1% Invitrogen 配方 3 L-Glutamine (200mM) 1% Invitrogen Formula 3
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen  lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen
20ng/m R&D bFGF  20ng/m R&D bFGF
1 Systems 1 Systems
20ng/m R&D20ng/m R&D
EGF 1 SystemsEGF 1 Systems
RPMI 1640 43ml Invitrogen 4(等同 FBS 5ml Invitrogen 于配方 HEPES ( 100X ) 0.5ml InvitrogenRPMI 1640 43ml Invitrogen 4 (equivalent to FBS 5ml Invitrogen in formula HEPES ( 100X ) 0.5ml Invitrogen
^ 丙酮酸 1 ( 100X ) 0.5ml Invitrogen ^ Pyruvic acid 1 ( 100X ) 0.5ml Invitrogen
L-glutamine ( 100X ) 0.5ml Invitrogen Antibiotic ( 100X ) 0.5ml Invitrogen  L-glutamine ( 100X ) 0.5ml Invitrogen Antibiotic ( 100X ) 0.5ml Invitrogen
20ng/m R&D  20ng/m R&D
Human bFGF  Human bFGF
1 Systems 1 Systems
20ng/m R&D 20ng/m R&D
Human EGF  Human EGF
1 Systems 多角的星状细胞 （胰岛干细胞或称为胰腺前体细胞） 进行 细月包免疫组化检测。 该细 JJ包的免疫组化染色按以下方法： 培养 细月包在 4。C 4%的多聚甲醛中固定 20分钟， 然后 0.01M PBS洗 三遍（培养细胞接种于涂有明胶的盖玻片上） ， 5%封闭用正常 羊血清室温下封闭 1小时， 然后 4。C一抗孵浴过夜， 0.01MPBS 洗三遍， 荧光二抗室温孵浴 2 小时， 0.01MPBS 洗三遍， 封片 后于 Nikon2000U 荧光显 ¾：镜下观察。 一抗和二抗的抗体同实 施例 1。 多角的星状细胞进行 RT-PCR扩增， RT-PCR分析采用以 下方法： 细包的总 RNA采用 TRIZOL法提取， 2 克的总 RNA被 逆转录为 cDNA， RT-PCR的反应体系由 50mM Tris-HCl ( pH 8.3 )、 50mM KCK 10 mM MgCl2、 lOmM DTT. 0.5mM亚精胺、 20U Rnasin、 0.5mM dNTP 、 0.5 μ g 01igo(dT)] 5、 50U SuperScriptTM II RT (厂家： Invitrogen ), 和无核醉水构成， 然 后反应体系在 42°C水浴 60分钟和 72°C水浴 1 分钟。然后通过 30-45 循环的 PCR扩增目标基因， PCR 的反应体系由 10mM Tris-HCK 50mM KC1、 0.1%Triton X-100、 1.75mM MgCl2、 0.4mM dNTP, 0.2 μ Μ有义和反义引物、 6.25U Taq DNA聚合酶、 以 及 3 μ g cDNA构成。 变性温度为 94 °C时间为 45秒， 退火温度 才艮据具体引物决定， 时间为 30秒， 延伸温度为 72°C时间为 7 分钟。 PCR产物通过 1.5%的凝胶电泳分离。 引物序列： 胰岛素上游引物： TCA CAC CTG GTG GAA GCT C (序列 19 ) , 下游引物： ACA ATG CCA CGC TTC TGC (序列 20 ) ( 179bp ) 1 Systems Multi-angled stellate cells (islet stem cells or pancreatic precursor cells) were subjected to fine-month immunohistochemistry. The immunohistochemical staining of the fine JJ package was as follows: Culture the fine monthly bag at 4. C 4% paraformaldehyde was fixed for 20 minutes, then washed three times with 0.01 M PBS (culture cells were seeded on gelatin-coated coverslips), and 5% blocked with normal goat serum for 1 hour at room temperature, then 4. C-antibiotic incubation was overnight, washed three times with 0.01 MPBS, incubated with fluorescent secondary antibody for 2 hours at room temperature, washed three times with 0.01 MPBS, and then observed under Nikon2000U fluorescence. The antibodies of the primary antibody and the secondary antibody were the same as in Example 1. Multi-angle stellate cells were subjected to RT-PCR amplification. The following methods were used for RT-PCR analysis: The fine-packed total RNA was extracted by TRIZOL method, 2 g of total RNA was reverse-transcribed into cDNA, and the RT-PCR reaction system was 50 mM Tris. -HCl (pH 8.3), 50 mM KCK 10 mM MgCl 2 , 10 mM DTT. 0.5 mM spermidine, 20 U Rnasin, 0.5 mM dNTP, 0.5 μg 01igo(dT) ] 5 , 50U SuperScript TM II RT (Manufacturer: Invitrogen) And consisting of non-nuclear drunk water, and then the reaction system was bathed in a water bath at 42 ° C for 60 minutes and a water bath at 72 ° C for 1 minute. The target gene was then amplified by PCR at 30-45 cycles. The PCR reaction system consisted of 10 mM Tris-HCK 50 mM KC1, 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 μ Μ sense and antisense primers. , 6.25 U Taq DNA polymerase, and 3 μg cDNA. The denaturation temperature is 94 °C for 45 seconds, and the annealing temperature is determined according to the specific primer. The time is 30 seconds, and the extension temperature is 72 °C. minute. The PCR product was separated by 1.5% gel electrophoresis. Primer sequence: Insulin upstream primer: TCA CAC CTG GTG GAA GCT C (SEQ ID NO: 19), downstream primer: ACA ATG CCA CGC TTC TGC (SEQ ID NO: 20) ( 179 bp )
Somatostatin上游引物： GTT TCT GCA GAA GTC TGG G (序歹 'J 21 ) , 下游引物： AGT TCT TGC AGC CAG CTT TG (序 歹' J 22 ) ( 223 bp ) Upstream Primer of Somatostatin: GTT TCT GCA GAA GTC TGG G (Sequence 歹 'J 21 ) , Downstream Primer: AGT TCT TGC AGC CAG CTT TG (Order 歹 ' J 22 ) ( 223 bp )
PDX-1上游引物： GGA TGA AGT CTA CCA AAG CTC ACG C (序歹 'J 23 ) , 下游引物： CCA GAT CTT GAT GTG TCT CTC GGT C (序歹 ll 24 ) ( 218bp ) 。 巢蛋白上游引物： AGA GGG GAA TTC CTG GAG (序列 25 ) 和 CTG AGG ACC AGG ACT CTC TA (序歹 'J 26 ) (496bp)。 PDX-1 upstream primer: GGA TGA AGT CTA CCA AAG CTC ACG C (Sequence 歹 'J 23 ), downstream primer: CCA GAT CTT GAT GTG TCT CTC GGT C (Serial ll 24 ) (218 bp). Nestin upstream primers: AGA GGG GAA TTC CTG GAG (sequence 25) and CTG AGG ACC AGG ACT CTC TA (sequence 歹 'J 26 ) (496 bp).
ABCG2上游引物： GGT CTC AGG AAG ACT TAT GT (序 列 27 ) , 下游引物： AAG GAG GTG GTG TAG CTG AT (序 歹' J 28 ) ( 323 bp ) 。 ABCG2 upstream primer: GGT CTC AGG AAG ACT TAT GT (sequence 27), downstream primer: AAG GAG GTG GTG TAG CTG AT (preface 歹 ' J 28 ) ( 323 bp ).
Isll上；斿引物： CTT AAA TTG GAC TCC TAG AT (序列 29 ) , 下游引物： GGA TTT GGA ATG GCA TGC GG (序列 30 ) ( 280bp ) 。 Isll; 斿 primer: CTT AAA TTG GAC TCC TAG AT (SEQ ID NO: 29), downstream primer: GGA TTT GGA ATG GCA TGC GG (sequence 30) (280 bp).
Glut2上游引物： TCC TGG CCT TTA CCC TGT TTA C(序 歹 'J 31 ) , 下游引物： CAG ACG GTT CCC TTA TTG TTT C (序 歹 'J 32 ) ( 209bp ) 。 β - actin上游引物： TGGCACCACACCTTCTACAATGAGC (序歹 'J 33 )，下游引物： GCACAGCTTCTCCTTAATGTCACGC (序歹 'J 34 ) ( 396bp ) 。 免疫组化结果显示： 这些多角形细胞为巢蛋白基因阳性细 胞， 如图 3B所示。 RT- PCR结果如图 3A中左侧的 PPC所示， 对于该多角形细胞， 下列基因表达为阳性： 巢蛋白， ABCG2, β -actin, 而下列基因表达为阴性: PDX- 1、 ISL-1、 Glut- 2、 胰 岛素基因、 生长激素 p制素基因 （Somatostatin ) 。 Glut2 upstream primer: TCC TGG CCT TTA CCC TGT TTA C (Sequence 歹 'J 31 ), downstream primer: CAG ACG GTT CCC TTA TTG TTT C (Sequence 歹 'J 32 ) (209 bp ). Β-actin upstream primer: TGGCACCACACCTTCTACAATGAGC (Sequence 歹 'J 33 ), downstream primer: GCACAGCTTCTCCTTAATGTCACGC (Sequence 歹 'J 34 ) (396bp). The results of immunohistochemistry showed that these polygonal cells were nestin gene positive cells, as shown in Figure 3B. The RT-PCR results are shown in the PPC on the left side of Figure 3A. For the polygonal cells, the following genes were positive: nestin, ABCG2, β-actin, and the following genes were negative: PDX-1, ISL-1 , Glut-2, insulin gene, growth hormone p-systemin gene (Somatostatin).
实施例 4 将胰岛干细胞培养成为脑腺内分泌细胞 将实施例 3 中得到的星形胰岛干细胞用 0.25%的胰酶将细 胞消化下来后， Hank's液中洗 1遍， 然后将细胞接种于含有培 养基 C的细菌培养 中 3天，得到前类胰岛团样结构（pre-ICC ), 图 2D。 3天后将培养基更换为培养基 D, 但仍然在细菌培养 中， 培养大约 4-6天， 得到 ICC结构的胰腺内分泌细胞。 以下为培养基 C和培养基 D的成分， 在实施例 4中, 我们 采用的具体配方都是配方 7 , 但是采用文中提到的其他配方也 可以同样得到胰月泉内分泌细月包。 另夕卜， 根据实验结果， 在含培养基 C (配方 7 ) 的培养皿 中的培养可以为 2-5天， 如图所示， 分别为培养 2天 （图 6A ) 和 5天（图 6B )得到的类胰岛团样结构。 根据实验结果， 在培 养基 D (配方 7 ) 中培养 4-15天均可以得到胰腺内分泌细胞， 如图所示， 分别为培养 10天 （图 6 C ) 和 15天 （图 6D ) 所得 到的胰腺内分泌细 。 培养基 C Example 4 Incubation of islet stem cells into brain gland endocrine cells The star islet stem cells obtained in Example 3 were digested with 0.25% trypsin, washed in Hank's solution, and then seeded in medium containing medium. In the bacterial culture of C for 3 days, a pre-islet-like structure (pre-ICC) was obtained, Fig. 2D. After 3 days, the medium was changed to medium D, but still in bacterial culture, cultured for about 4-6 days to obtain pancreatic endocrine cells of ICC structure. The following are the components of medium C and medium D. In Example 4, the specific formulation we used was Formulation 7, but the other formulas mentioned in the text were also used to obtain the endocrine sac. In addition, according to the experimental results, the culture in the culture dish containing the medium C (formulation 7) may be 2-5 days, as shown in the figure, respectively, for 2 days of culture (Fig. 6A) and 5 days (Fig. 6B). The resulting islet-like structure. According to the experimental results, pancreatic endocrine cells can be obtained by culturing for 4-15 days in medium D (formulation 7), as shown in the figure, for 10 days (Fig. 6 C ) and 15 days (Fig. 6D ). The pancreas is secreted finely. Medium C
成分 比例 厂家 Composition ratio manufacturer
DMEM (无糖 )或 （ 3mmol/l葡萄糖） 48% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 48% Invitrogen
F12 48% InvitrogenF12 48% Invitrogen
B27 2% InvitrogenB27 2% Invitrogen
5% BSA 1% Invitrogen 配方 1 20ng 5% BSA 1% Invitrogen Formula 1 20ng
bFGF Invitrogen  bFGF Invitrogen
/ml  /ml
20ng  20ng
EGF Invitrogen  EGF Invitrogen
/ml  /ml
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen
DMEM (无糖 )或 （ 3mmol/l葡萄糖） 48% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 48% Invitrogen
F12 48% InvitrogenF12 48% Invitrogen
B27 2% SigmaB27 2% Sigma
10% BSA 1% Invitrogen 配方 2 20ng 10% BSA 1% Invitrogen Formula 2 20ng
bFGF Invitrogen  bFGF Invitrogen
/ml  /ml
20ng  20ng
EGF Invitrogen  EGF Invitrogen
/ml  /ml
lOOOOu青霉素 -lOOOOug链霉素 1% 00 Invitrogen lOOOOu penicillin - lOOOOug streptomycin 1% 00 Invitrogen
DMEM (无糖 )或 （ 3mmol/l葡萄糖） 48% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 48% Invitrogen
F12 InvitrogenF12 Invitrogen
B27 2% SigmaB27 2% Sigma
20% BSA 1% Invitrogen 配方 3 20ng 20% BSA 1% Invitrogen Formula 3 20ng
bFGF Invitrogen  bFGF Invitrogen
/ml  /ml
20ng  20ng
EGF Invitrogen  EGF Invitrogen
/ml  /ml
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen
DMEM (无糖 )或 （ 3mmol/l葡萄糖） 49% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 49% Invitrogen
F12 49% InvitrogenF12 49% Invitrogen
ITS lg/L SigmaITS lg/L Sigma
5% BSA 1% Invitrogen 配方 4 20ng 5% BSA 1% Invitrogen Formula 4 20ng
bFGF Invitrogen  bFGF Invitrogen
/ml  /ml
20ng  20ng
EGF Invitrogen  EGF Invitrogen
/ml  /ml
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen 配方 DMEM (无糖 )或 （ 3mmol/l葡萄糖） 49% Invitrogen  lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen formula DMEM (no sugar) or (3mmol/l glucose) 49% Invitrogen
F12 49% Invitrogen F12 49% Invitrogen
ITS lg/L Sigma 10% BSA 1% In vitro gen ITS lg/L Sigma 10% BSA 1% In vitro gen
20ng  20ng
bFGF Invitrogen  bFGF Invitrogen
/ml  /ml
20ng  20ng
EGF Invitrogen  EGF Invitrogen
/ml  /ml
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen DMEM (无糖）或 （ 3mmol/l葡萄糖） 49% Invitrogen F12 49% Invitrogen ITS lg/L Sigma lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen DMEM (no sugar) or (3mmol/l glucose) 49% Invitrogen F12 49% Invitrogen ITS lg/L Sigma
20% BSA 1% Invitrogen 配方 6 20ng 20% BSA 1% Invitrogen Formula 6 20ng
bFGF Invitrogen  bFGF Invitrogen
/ml  /ml
20ng  20ng
EGF Invitrogen  EGF Invitrogen
/ml  /ml
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen  lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen
48m  48m
DMEM/F12 ( 5.6mM 葡萄糖） Invitrogen  DMEM/F12 (5.6mM glucose) Invitrogen
1  1
B27 ( 50 x ) lml Invitrogen  B27 ( 50 x ) lml Invitrogen
0.5  0.5
7.5% BSA Invitrogen  7.5% BSA Invitrogen
ml  Ml
配方 7 20ng Formula 7 20ng
bFGF Invitrogen  bFGF Invitrogen
/ml  /ml
20ng  20ng
EGF Invitrogen  EGF Invitrogen
/ml  /ml
0.5  0.5
Invitrogen Invitrogen
Antibiotics( 00 ) ml 培养基 D Antibiotics( 00 ) ml medium D
成分 比例 厂家 Composition ratio manufacturer
DMEM (无糖 )或 （ 3mmol/l葡萄糖） 48% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 48% Invitrogen
F12 48% InvitrogenF12 48% Invitrogen
B27 2% InvitrogenB27 2% Invitrogen
5% BSA 1% Invitrogen 5% BSA 1% Invitrogen
10m  10m
烟酰胺 Sigma  Niacinamide Sigma
M  M
配方 1 10η  Formula 1 10η
GLP-1 ( 7-36 amide ) Sigma  GLP-1 ( 7-36 amide ) Sigma
M  M
10ng  10ng
HGF R&D Systems  HGF R&D Systems
/ml  /ml
500p  500p
Betacellulin R&D Systems  Betacellulin R&D Systems
M  M
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen 配方 2 DMEM (无糖）或 （ 3mmol/l葡萄糖） 48% Invitrogen F12 48% InvitrogenlOOOOu penicillin-lOOOOug streptomycin 1% Invitrogen Formula 2 DMEM (no sugar) or (3mmol/l glucose) 48% Invitrogen F12 48% Invitrogen
B27 2% InvitrogenB27 2% Invitrogen
10% BSA - 1% Invitrogen 10% BSA - 1% Invitrogen
10m  10m
烟酸胺 Sigma  Niacinamine Sigma
M  M
10η  10η
GLP-1 ( 7-36 amide ) Sigma  GLP-1 ( 7-36 amide ) Sigma
M  M
lOng  lOng
HGF R&D Systems  HGF R&D Systems
/ml  /ml
500p  500p
Betacellulin R&D Systems  Betacellulin R&D Systems
M  M
lOOOOu青霉素 - lOOOOug链霉素 1% Invitrogen lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen
DMEM (无糖 ) 或 （ 3mmol/l葡萄糖） 48% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 48% Invitrogen
F12 48% InvitrogenF12 48% Invitrogen
B27 2% InvitrogenB27 2% Invitrogen
20% BSA 1% Invitrogen 20% BSA 1% Invitrogen
10m  10m
烟酰胺 Sigma  Niacinamide Sigma
M  M
配方 3 10η Formula 3 10η
GLP-1 ( 7-36 amide ) Sigma  GLP-1 ( 7-36 amide ) Sigma
M  M
lOng  lOng
HGF R&D Systems  HGF R&D Systems
/ml  /ml
500p  500p
Betacellulin R&D Systems  Betacellulin R&D Systems
M  M
lOOOOu青霉素 - lOOOOug链霉素 1% Invitrogen lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen
DMEM (无糖 ) 或 （ 3mmol/l葡萄糖） 49% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 49% Invitrogen
F12 49% InvitrogenF12 49% Invitrogen
ITS lg/L SigmaITS lg/L Sigma
5% BSA 1% Invitrogen 5% BSA 1% Invitrogen
10m  10m
烟酰胺 Sigma  Niacinamide Sigma
M  M
配方 4 10η Formula 4 10η
GLP-1 ( 7-36 amide ) Sigma  GLP-1 ( 7-36 amide ) Sigma
M  M
lOng  lOng
HGF R&D Systems  HGF R&D Systems
/ml  /ml
500p  500p
Betacellulin R&D Systems  Betacellulin R&D Systems
M  M
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen
DMEM (无糖 ) 或 （ 3mmol/l葡萄糖） 49% InvitrogenDMEM (no sugar) or (3mmol/l glucose) 49% Invitrogen
F12 49% InvitrogenF12 49% Invitrogen
ITS lg/L SigmaITS lg/L Sigma
10%BSA 1% Invitrogen 10% BSA 1% Invitrogen
10m  10m
烟酰胺 Sigma M On Niacinamide Sigma M On
GLP-1 ( 7-36 amide ) Sigma  GLP-1 ( 7-36 amide ) Sigma
Ong  Ong
HGF R&D Systems  HGF R&D Systems
/ml  /ml
500p  500p
Betacellulin R&D Systems  Betacellulin R&D Systems
M  M
lOOOOu青霉素 - lOOOOug链霉素 % Invitrogen DMEM (无糖）或（ 3mmol/l葡萄糖） 49% Invitrogen F12 49% Invitrogen ITS lg/L Sigma lOOOOu penicillin - lOOOOug streptomycin % Invitrogen DMEM (no sugar) or (3mmol/l glucose) 49% Invitrogen F12 49% Invitrogen ITS lg/L Sigma
20% BSA 1% Invitrogen20% BSA 1% Invitrogen
On  On
烟醜胺 Sigma  Smoke ugly amine Sigma
M  M
配方 6  Formula 6
10η  10η
GLP-1 ( 7-36 amide ) Sigma  GLP-1 ( 7-36 amide ) Sigma
M  M
lOng  lOng
HGF R&D Systems  HGF R&D Systems
/ml  /ml
5000p  5000p
Betacellulin R&D Systems  Betacellulin R&D Systems
M  M
lOOOOu青霉素 -lOOOOug链霉素 1% Invitrogen DMEM/F12 ( 5.6mM葡萄糖） 48ml Invitrogen B27 lml Invitrogen  lOOOOu penicillin - lOOOOug streptomycin 1% Invitrogen DMEM/F12 (5.6mM glucose) 48ml Invitrogen B27 lml Invitrogen
0.5m  0.5m
7.5% BSA(100 χ ) Invitrogen  7.5% BSA (100 χ ) Invitrogen
1  1
10m  10m
配方 7 烟 @¾胺 Sigma  Formulation 7 Smoke @3⁄4amine Sigma
M  M
(等同 10η  (equivalent to 10η
于配方 GLP-1 ( 7-36 amide ) Sigma  In formula GLP-1 ( 7-36 amide ) Sigma
M  M
1 ) lOng  1) lOng
HGF R&D Systems  HGF R&D Systems
/ml  /ml
500p  500p
Betacellulin R&D Systems  Betacellulin R&D Systems
M  M
0.5m  0.5m
Antibiotics(100 x ) Invitrogen  Antibiotics(100 x ) Invitrogen
1 对 ICC结构的胰腺内分泌细胞进行细胞免疫组化检测。 免 疫组乜染色按以下方法： ICC在 4°C 4%的多聚甲醛中固定 1小 时，然后 0.01M PBS洗三遍（ ICC接种于涂有明胶的盖玻片上）， 5% †闭用正常羊血清室温下^) "闭 1小时， 然后 4。C一抗孵育过 夜， 0.01MPBS洗三遍， 荧光二抗室温孵育 2小时， 0.01MPBS 洗三遍， 封片后于 Nikon2000U 荧光显 镜下观察。 一抗和二 抗的抗体同实施例 1。 ICC 结构的月夷腺内分泌细胞进行 RT-PCR扩增。 RT- PCR 分析采用以下方法： 细胞的总 R A采用 TRIZOL法提取， 2 4敖克的总 RNA被 逆转录为 cDNA , RT-PCR 的反应体系由 50mM Tris-HCl ( ρΗ8·3 )、 50mM KCl、 10mM MgCl2、 10mM DTT、 0.5mM亚 精胺、 20U Rnasin、 0.5mM dNTP、 0.5 μ g 01igo(dT)15、 50U Superscript™ II RT ( Invitrogen )、 和无核酶水构成， 然后反应 体系在 42。C水浴 60分钟和 72 °C水浴 15分钟。 然后通过 30-45 循环的 PCR扩增目标基因，PCR的反应体系由 10mM Tris-HCK 50mM KCK 0.1%Triton X-100, 1.75mM MgCl2、 0.4mM dNTP、 0.2 μ M有义和反义引物、 6.25U Taq DNA聚合醉、 以及 3 μ g cDNA构成。 变性温度为 94°C时间为 45秒， 退火温度根据具 体引物决定， 时间为 30秒， 延伸温度为 72°C时间为 7分钟。 PCR产物通过 1.5%的凝胶电泳分离。 1 Cellular immunohistochemistry was performed on pancreatic endocrine cells of ICC structure. The immunization group was stained as follows: ICC was fixed in 4% paraformaldehyde for 4 hours at 4 ° C, then washed three times with 0.01 M PBS (ICC was inoculated on gelatin-coated coverslips), and 5% † was used normally. Sheep serum was incubated at room temperature for 2 hours, then incubated overnight with 4 C primary antibody, washed three times with 0.01 M PBS, incubated with fluorescent secondary antibody for 2 hours at room temperature, washed three times with 0.01 M PBS, and mounted under Nikon 2000 U fluorescence microscope. The antibodies of primary and secondary antibodies are the same as in Example 1. The ICC-structured squamous cell endocrine cells were subjected to RT-PCR amplification. The following methods were used for RT-PCR analysis: Total RA of cells was extracted by TRIZOL method, and total RNA of 24 g was reverse-transcribed into cDNA. The reaction system of RT-PCR consisted of 50 mM Tris-HCl (ρΗ8·3), 50 mM KCl, 10mM MgCl 2 , 10mM DTT, 0.5mM spermidine, 20U Rnasin, 0.5mM dNTP, 0.5 μg 01igo(dT) 15 , 50U SuperscriptTM II RT ( Invitrogen ), and nuclease-free water, then the reaction system is at 42 . C water bath for 60 minutes and 72 °C water bath for 15 minutes. The target gene was then amplified by PCR at 30-45 cycles. The PCR reaction system consisted of 10 mM Tris-HCK 50 mM KCK 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 μM sense and antisense primers, 6.25 U Taq DNA polymerization drunk, and 3 μ g cDNA. The denaturation temperature was 94 ° C for 45 seconds, the annealing temperature was determined according to the specific primer, the time was 30 seconds, and the extension temperature was 72 ° C for 7 minutes. The PCR product was separated by 1.5% gel electrophoresis.
RT-PCR分析结果显示， ICC与 PPC比较， ICC的巢蛋白 基因、 ABCG2基因表达减弱或消失，而胰岛相关基因开始表达， 胰岛素、 肢高血糖素、 生长抑素和 PDX-1、 ISL1、 GLUT2等基 因表达呈阳性， 见图 3A。 免疫组化检测显示， ICCs 中胰岛素 阳性细胞主要分布于 ICC的中央而胰高血糖素阳性细胞主要分 布于 ICC的周边， 见图 3C。 胰岛素和 C肽含量的测定 镜下 ^选 200个正常胰岛、 Pre-ICC (进入培养基 D之前 的） 或 ICC (指经培养基 D分化后的 ICC ) , 放入 0.5ml 的小 离心管中， 用无糖 DMEM洗两遍， 每遍 5分钟， 然后用 30mM 的葡萄糖于 37°C (02/C02, 95:5)孵育 90分钟， 然后再收集上清 用 ACTIVE Insulin ELISA 和 ACTIVE C-Peptide of Insulin ELISA试剂盒分析测定， 结果见图 4。 图 4显示 Pre-ICCs、 ICCs和正常胰岛团在 30mM高糖刺激 下胰岛素（图 4A)和 C肽（图 4B)的分泌情况。 胰岛素的分泌情况是（μ Γϋ/ml): pre-ICCs, 3.47 ± 1.55; ICC, 45.02 ± 2.94; 月夷岛团， 330.63 ± 31.70。 RT-PCR analysis showed that ICC compared with PPC, ICC nestin gene, ABCG2 gene expression decreased or disappeared, and islet related genes began to express, insulin, limb glucagon, somatostatin and PDX-1, ISL1, GLUT2 Isogenic expression was positive, see Figure 3A. Immunohistochemistry showed that insulin-positive cells in ICCs were mainly distributed in the center of ICC and glucagon-positive cells were mainly distributed in the periphery of ICC, as shown in Figure 3C. Determination of insulin and C-peptide content Select 200 normal islets, Pre-ICC (before entering medium D) or ICC (refer to ICC after medium D differentiation), and place in a 0.5 ml small centrifuge tube. Wash twice with sugar-free DMEM for 5 minutes each time, then incubate with 30 mM glucose at 37 ° C (0 2 /C0 2 , 95:5) for 90 minutes, then collect the supernatant with ACTIVE Insulin ELISA and ACTIVE C -Peptide of Insulin The ELISA kit was analyzed and the results are shown in Figure 4. Figure 4 shows Pre-ICCs, ICCs and islets normal insulin (FIG. 4A) at high glucose 30mM and C-peptide (Fig. 4 B) of the secretion. The secretion of insulin was (μ Γϋ / ml): pre-ICCs, 3.47 ± 1.55; ICC, 45.02 ± 2.94; Yueyi Island, 330.63 ± 31.70.
C肽的分泌情况是 (ng/ml): pre-ICC, 0 ± 0; ICC, 2.19士 0.40; 胰岛团， 19.85 ± 2.34。 实施例 5糖尿病动物模型胰腺组织与正常动物胰腺组织 各个基因转录水平比较的检测 The secretion of C peptide was (ng/ml): pre-ICC, 0 ± 0; ICC, 2.19 ± 0.40; islet group, 19.85 ± 2.34. Example 5 Detection of transcript levels of various genes in pancreatic tissue of pancreatic tissue and normal animal pancreas
RT-PCR分析采用以下方法： 细胞或组织的总 RNA采用 TRIZOL法提取， 2 克的总 RNA被逆转录为 cDNA, RT- PCR的反应体系由 50mM Tris-HCl ( pH 8.3 )、 50mM KCK 10 mM MgCl2、 10 mM DTT、 0.5 mM 亚 4青胺、 20U Rnasin, 0.5mM dNTP、 0.5 μ g 01igo(dT)15、 50 U Superscript™ II RT (厂家： Invitrogen )、 和无核酶水构成， 然 后反应体系在 42°C水浴 60分钟和 72°C水浴 15分钟。然后通过 30-45 循环的 PCR扩增目标基因， PCR 的反应体系由 10mM Tris-HCK 50mM KC1、 0.1%Triton X-100、 1.75mM MgCl2、 0.4mM dNTP、 0.2 μ Μ有义和反义引物 ( sense and antisense primer )、 6.25U Taq DNA聚合酶、 以及 3 μ gcDNA构成。 变性温度为 94 °C时间为 45秒， 退火温度才艮据具体引物决定， 时间为 30秒， 延伸温度为 72 时间为 7分钟。 每个基因的 PCR扩增的标准 曲线 （即被扩增的 DNA 片段的数量与循环圈数关系的曲线） 用 Real-time PCR(MJ Research, CFD-3220)的方法建立。 为了比 较各个基因的转录水平， 各个 PCR 所采用的是循环圈数是 Real-time PGR标准曲线上的最大循环圈数。 PCR产物通过 1.5% 的凝胶电泳分离。 引物序列： 胰岛素上游引物： GCAGCCTTTGTGAACCAACAC (序列 35 ) , 下游引物： CCCCGCACACTAGGTAGAGA (序列 36 ) ( 67bp ) The following methods were used for RT-PCR analysis: Total RNA of cells or tissues was extracted by TRIZOL method, 2 g of total RNA was reverse-transcribed into cDNA, and RT-PCR reaction system consisted of 50 mM Tris-HCl (pH 8.3), 50 mM KCK 10 mM. MgCl 2 , 10 mM DTT, 0.5 mM sub-4-cyanamine, 20 U Rnasin, 0.5 mM dNTP, 0.5 μg 01igo(dT) 15 , 50 U SuperscriptTM II RT (manufacturer: Invitrogen), and nuclease-free water, then The reaction system was subjected to a water bath at 42 ° C for 60 minutes and a 72 ° C water bath for 15 minutes. The target gene was then amplified by PCR at 30-45 cycles. The PCR reaction system consisted of 10 mM Tris-HCK 50 mM KC1, 0.1% Triton X-100, 1.75 mM MgCl 2 , 0.4 mM dNTP, 0.2 μ Μ sense and antisense primers. (Sense and antisense primer), 6.25 U Taq DNA polymerase, and 3 μg cDNA. The denaturation temperature was 94 °C for 45 seconds, and the annealing temperature was determined according to the specific primer, the time was 30 seconds, and the extension temperature was 72 minutes for 7 minutes. The standard curve of PCR amplification of each gene (i.e., the relationship between the number of amplified DNA fragments and the number of cycles) was established by Real-time PCR (MJ Research, CFD-3220). In order to compare the transcription levels of individual genes, the number of cycles used in each PCR is The maximum number of cycles on the Real-time PGR standard curve. The PCR product was separated by 1.5% gel electrophoresis. Primer sequence: Insulin upstream primer: GCAGCCTTTGTGAACCAACAC (SEQ ID NO: 35), downstream primer: CCCCGCACACTAGGTAGAGA (SEQ ID NO: 36) (67bp)
Glucagon上游引物： ATTCACAGGGCACATTCACC (序列 37 ) , 下游引物： AACAATGGCGACCTCTTCTG (序列 38 ) ( 260bp ) 。 Glucagon upstream primer: ATTCACAGGGCACATTCACC (sequence 37), downstream primer: AACAATGGCGACCTCTTCTG (sequence 38) (260bp).
Somatostatin上游引物： GCTGCTGTCTGAACCCAAC (序 歹1 39 ) , 下游引物： CGTTCTCGGGGTGCCATAG (序列 40 ) ( 138bp ) Somatostatin upstream primer: GCTGCTGTCTGAACCCAAC (Sequence 391 39), downstream primer: CGTTCTCGGGGTGCCATAG (SEQ ID NO: 40) ( 138bp )
PDX-1 上游引物： TGATACTGGATTGGCGTTGT (序列 41 ) , 下游引物： GCATCAATTTCACGGGATCT (序列 42 ) ( 270bp ) 巢蛋白上游引物： AAGAGCTGGAGGGCGTGGTG (序列 43 ) 和 TCCTGATAGCCGCGCACTG (序歹 'J 44 ) ( 328bp ) PDX-1 upstream primer: TGATACTGGATTGGCGTTGT (SEQ ID NO: 41), downstream primer: GCATCAATTTCACGGGATCT (SEQ ID NO: 42) (270bp) Nestin upstream primer: AAGAGCTGGAGGGCGTGGTG (sequence 43) and TCCTGATAGCCGCGCACTG (sequence 歹 'J 44 ) (328bp)
ABCG2 上游引物： GGCCTCAGGAAGACTTATGT (序列 45 ) , 下游引物： AAGGAGGTGGTGTAGCTGAT (序歹 'J 46 ) ( 342bp ) ABCG2 upstream primer: GGCCTCAGGAAGACTTATGT (sequence 45), downstream primer: AAGGAGGTGGTGTAGCTGAT (prologue 'J 46 ) ( 342bp )
Isll上游引物： TGTTTGAAATGTGCGGAGTG (序歹 'J 47 ) , 下游引物： GTTCTTGCTGAAGCCGATG (序歹 'J 48 ) ( 144bp ) Glut2上 i 引物： TTGCTGGAAGAAGCATATCAGG (序列 49 ) , 下游引物： TGACTAATAAGAATGCCCGTGAC (序列 50 ) ( 148bp ) β -actin上游引物： TGGCACCACACCTTCTACAATGAGC (序列 51 ) , 下游引物： GCACAGCTTCTCCTTAATGTCACGC (序列 52 ) ( 396bp ) 。 本发明动物模型胰腺组织的 RT-PCR分析结果显示如图 7 所示， 其中左侧的是正常猴的胰腺组织细胞的结果， 右侧是糖 尿病猴的胰腺组织的结果。 如图所示， 糖尿病动物胰腺组织中 胰岛素基因 （Insulin )表达减少， 胰高血糖素基因 （Glucagon ) 表达增加和生长抑素基因 （Somatostatin ) 表达无明显改变， PDX-1 基因表达无明显改变， β -actin基因表达无明显改变， 干细胞标志巢蛋白基因表达有所增加， Glut-2 基因表达减少。 这说明在本发明使用的模型中， 虽然胰岛 β细胞被严重破坏， 但 在残存的胰岛内， 巢蛋白阳性的胰腺干细胞仍然存在于糖尿病 动物的胰泉组织中。 实施例 6对多角的星状 干细胞与诱导分化后的 ICC 各个基因转录水平的检测 对实施例 3的多角的星状胰腺干细胞与实施例 4的诱导分 化后的 ICC 各个基因转录水平的检测。 细胞总 RNA 采用 TRIZOL法提取， 2 ^:克的总 RNA被逆转录为 cDNA, RT-PCR 的反应体系由 50mM Tris-HCl ( pH 8.3 )、 50mM KC1、 10 mM MgCl2、 10 mM DTT、 0.5 mM亚精胺、 20U Rnasin, 0.5mM dNTP、 0.5 μ g 01igo(dT)15、 50 U Superscript™ II RT (厂家： Invitrogen )、 和无核酶水构成， 然后反应体系在 42°C水浴 60分钟和 72°C水 浴 15分钟。 然后通过 30-45循环的 PCR扩增目标基因， PCR 的反应体系由 10mM Tris-HCl、 50mM KCl、 0.1%Triton X-100 1.75mM MgCl2、 0.4mM dNTP 0.2 μ M有义和反义引物（ sense and antisense primer )、 6.25U Taq DNA聚合錄、 以及 3 μ gcDNA 构成。 变性温度为 94 °C时间为 45秒， 退火温度才艮据具体引物 决定， 时间为 30秒， 延伸温度为 72 °C时间为 7分钟。 每个基 因的 PCR扩增的标准曲线 （即被扩增的 DNA片段的数量与循 环圈数关系的曲线） 用 Real- time PCR(MJ Research, CFD-3220) 的方法建立。 为了比较各个基因的转录水平， 各个 PCR所采用 的是循环圈数是 Real-time PCR标准曲线上的最大循环圈数。 PCR产物通过 1.5%的;疑胶电泳分离 引物序列： 胰岛素上游引物： GCAGCCTTTGTGAACCAACAC (序列 53 ) , 下游引物： CCCCGCACACTAGGTAGAGA (序列 54 ) ( 67bp ) Isll upstream primer: TGTTTGAAATGTGCGGAGTG (prologue 'J 47 ), downstream primer: GTTCTTGCTGAAGCCGATG (prologue 'J 48 ) ( 144bp ) Glut2 on i primer: TTGCTGGAAGAAGCATATCAGG (SEQ ID NO: 49), downstream primer: TGACTAATAAGAATGCCCGTGAC (SEQ ID NO: 50) (148 bp) β-actin upstream primer: TGGCACCACACCTTCTACAATGAGC (SEQ ID NO: 51), downstream primer: GCACAGCTTCTCCTTAATGTCACGC (SEQ ID NO: 52) (396bp). The results of RT-PCR analysis of pancreatic tissue of the animal model of the present invention are shown in Fig. 7, in which the left side is the result of pancreatic tissue cells of normal monkeys, and the right side is the result of pancreatic tissue of diabetic monkeys. As shown in the figure, the expression of insulin gene (Insulin) in pancreatic tissue of diabetic animals decreased, the expression of glucagon gene (Glucagon) increased and the expression of somatostatin gene did not change significantly, and the expression of PDX-1 gene did not change significantly. There was no significant change in the expression of β-actin gene, the expression of nestin gene was increased in stem cells, and the expression of Glut-2 gene was decreased. This indicates that in the model used in the present invention, although islet β cells are severely damaged, nestin-positive pancreatic stem cells are still present in the pancreatic tissue of diabetic animals in the remaining islets. Example 6 Detection of transcriptional levels of individual genes of ICCs after multi-angled stem cells and induced differentiation The detection of the transcriptional levels of individual genes of ICCs of the multi-angled stellate pancreatic stem cells of Example 3 and the induced differentiation of Example 4 was carried out. Total cellular RNA was extracted by TRIZOL method, 2 ^: gram of total RNA was reverse transcribed into cDNA, and RT-PCR reaction system consisted of 50 mM Tris-HCl (pH 8.3 ), 50 mM KC1, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM spermidine, 20 U Rnasin, 0.5 mM dNTP, 0.5 μg 01igo(dT) 15 , 50 U SuperscriptTM II RT (manufacturer: Invitrogen), and nuclease-free water, then the reaction system was bathed at 42 ° C for 60 minutes And a 72 ° C water bath for 15 minutes. The target gene is then amplified by PCR at 30-45 cycles, PCR The reaction system consists of 10 mM Tris-HCl, 50 mM KCl, 0.1% Triton X-100 1.75 mM MgCl 2 , 0.4 mM dNTP 0.2 μM sense and antisense primer, 6.25 U Taq DNA, and 3 μ g cDNA. The denaturation temperature was 94 °C for 45 seconds, and the annealing temperature was determined according to the specific primer, the time was 30 seconds, and the extension temperature was 72 °C for 7 minutes. The standard curve of PCR amplification of each gene (i.e., the relationship between the number of amplified DNA fragments and the number of cycles) was established by Real-time PCR (MJ Research, CFD-3220). In order to compare the transcription levels of individual genes, the number of loops used for each PCR is the maximum number of loops on the Real-time PCR standard curve. The PCR product was passed through 1.5%; the primer sequence was separated by electrophoresis: insulin upstream primer: GCAGCCTTTGTGAACCAACAC (sequence 53), downstream primer: CCCCGCACACTAGGTAGAGA (sequence 54) (67bp)
Somatostatin上游引物： GCTGCTGTCTGAACCCAAC (序 列 55 ) ， 下游引物： CGTTCTCGGGGTGCCATAG (序歹 'j 56 ) ( 138bp ) 。 Somatostatin upstream primer: GCTGCTGTCTGAACCCAAC (SEQ ID NO: 55), downstream primer: CGTTCTCGGGGTGCCATAG (Sequence 歹 'j 56 ) (138bp).
PDX- 1 上游引物： TGATACTGGATTGGCGTTGT (序列 57 ) , 下游引物: GCATCAATTTCACGGGATCT (序列 58 ) ( 270bp ) 巢蛋白上游引物： A AGAGCTGGAGGGCGTGGTG (序列 PDX-1 upstream primer: TGATACTGGATTGGCGTTGT (SEQ ID NO: 57), downstream primer: GCATCAATTTCACGGGATCT (SEQ ID NO: 58) (270bp) Nestin upstream primer: A AGAGCTGGAGGGCGTGGTG (sequence
ABCG2上游引物： GGCCTCAGGAAGACTTATGT (序列 61 ) , 下游引物： AAGGAGGTGGTGTAGCTGAT (序歹 'J 62 ) ( 342b ) Isll上游引物： TGTTTGAAATGTGCGGAGTG (序歹 'J 63 ) , 下 游引物： GTTCTTGCTGAAGCCGATG (序列 64 ) ： ( 144bp ) ABCG2 upstream primer: GGCCTCAGGAAGACTTATGT (SEQ ID NO: 61), downstream primer: AAGGAGGTGGTGTAGCTGAT (Sequence 歹 'J 62 ) ( 342b ) Isll upstream primer: TGTTTGAAATGTGCGGAGTG (prologue 'J 63 ), downstream primer: GTTCTTGCTGAAGCCGATG (sequence 64) : ( 144bp )
Glut2上游引物： TTGCTGGAAGAAGCATATCAGG (序列Glut2 upstream primer: TTGCTGGAAGAAGCATATCAGG (sequence
65 ) , 下游引物： TGACTAATAAGAATGCCCGTGAC (序列65) , downstream primer: TGACTAATAAGAATGCCCGTGAC (sequence
66 ) ( 148bp ) 。 β -actin上游引物： TGGCACCACACCTTCTACAATGAGC (序列 67 ) ,下游引物: GCACAGCTTCTCCTTAATGTCACGC (序歹 'J 68 ) ( 396bp ) 。 66) (148bp). Β-actin upstream primer: TGGCACCACACCTTCTACAATGAGC (SEQ ID NO: 67), downstream primer: GCACAGCTTCTCCTTAATGTCACGC (Sequence 歹 'J 68 ) (396bp).
RT-PCR结果如图 8 所示 ICC与 PPC比较， ICC的巢蛋白 基因、 ABCG2基因表达减弱或消失，而胰岛相关基因开始表达， 胰岛素、 胰高血糖素、 生长抑素和 PDX-1、 GLUT2等基因表达 呈阳性。 实施例 7 正常胰岛和诱导分化得到的 ICC在不同刺激物 的作用下胰岛素和 C肽分泌量的测定 镜下分别挑选 200个正常肢岛或来自实施例 4的 ICC, 放 入 0.5ml的小离心管中， 用无糖 DMEM洗两遍， 每遍 5分钟， 然后分别用 200ul含有不同浓度葡萄糖（OmM, 5.6mM 或者 16.7mM ) 的 DMEM或者 200ul含有 lOmML-精氨酸和不同浓 度葡萄糖（ OmM, 5.6mM或者 16.7mM )的 DMEM, 37°C( 02/C02， 95:5 )孵育 90分钟， 然后再收集上清用超敏感的胰岛素 ELISA 和超敏感的 C- Peptide腹岛素 ELISA试剂盒（ Mercodia, Uppsala, Sweden )分析测定， 结果见图 9。 图 9结果显示 当 ICC 和成体胰岛在不同浓度的葡萄糖或 在同时含有 1 OmM L-精氨酸的刺激下， 胰岛素（图 9A1、 9A2 ) 和 C-肽（图 9B1、 9B2 ) 的分泌情况如下： 在不同浓度葡萄糖 的刺激下， 胰岛素分泌量分别为 （mIUx lO-6/min/ICC or islet) ： 在 OmM 浓度的葡萄糖刺激下：正常成体胰岛的胰岛素分泌量为 0.2174±0.0632, ICC为 0.0561±0.0258； 在 5.6mM浓度的葡萄糖 刺激下： 成体胰岛的胰岛素分泌量为 0.9305±0.0896, ICC 为 0.0845±0.0339； 在 16.7mM 浓度的葡萄糖刺激下： 成体跌岛的 胰岛素分泌量为 2.0907±0.2644, ICC为 0.1謝 ±0.0542; C-肽的 分泌量为 (pmolx l0-6/min/islet or ICC): 在 OmM 浓度的葡萄糖 刺激下：成体胰岛的 C-肽分泌量为 4.441±1.218, ICC 为 0.0585±0.1013； 在 5.6mM浓度的葡萄糖刺激下：成体胰岛的 C- 肽分泌量为 7.3336±1.9997, ICC为 0.1295±0.1323； 在 16.7mM 浓度的葡萄糖刺激下： 成体胰岛的 C-肽分泌量为 9.4841±3.3433, ICC为 0.5423±0.1382.当在不同浓度的葡萄糖和 10mML4青氨酸 同时刺激下， 胰岛素的分泌量为 （mlUx lO— 6/min/ICC or islet): 在 OmM的葡萄糖和 10mML-精氨酸同时刺激下,成体胰岛的胰 岛素分泌量为 0.6003±0·0419, ICC为 0.0641±0.0279； 在 5.6mM 的葡萄糖和 10mML-精氨酸同时刺激下， 成体胰岛的胰岛素分 泌量为 2.7675±0.8867, ICC为 0.1809±0.0211；在的 16.7mM葡萄 糖和 lOmML-精氨酸同时刺激下， 成体胰岛的胰岛素分泌量为 6.1731±2.4574, ICC 为 0.2677±0.0792； C-肽的分泌量为 (pmolx l0"6/min/islet or ICC) 在 OmM的葡萄糖和 10mML4青氨 酸同时刺激下,成体胰岛的 C-肽分泌量为 5.5077±1.2808, ICC为 0.2984±0.0301,在 5.6mM的葡萄糖和 lOmML-精氨酸同时刺激 下，成体胰岛的 C-肽分泌量为 7.9897±0.4765, ICC 为 0.4901±0.1382，在 16.7mM的葡萄糖和 lOmML-精氨酸同时刺激 下，成体胰岛的 C-肽分泌量为 12.6138±2.8141, ICC 为 0·6295±0·2471。 结果显示， 根据本发明的方法， 从糖尿病动物的胰腺细胞 可以获得的 ICC结构的胰腺内分泌细胞， 该胰腺内分泌细胞可 以分泌胰岛素和 C肽。 该体外培养的胰腺内分泌细胞可以同体 或者异体移植给糖尿病动物， 使糖尿病动物可以摆脱胰岛素注 射的依赖。 并且解决了异体移植的排斥反应和千细胞来源不足 的问题。 另外， 本发明所提及的引物序列是由人工合成的 （可 由上海生工合成）。 以上所述仅为本发明的优选实施例而已， 并不用于限制本 发明， 对于本领域的技术人员来说， 本发明可以有各种更改和 变化。 凡在本发明的精神和原则之内， 所作的任何修改、 等同 替换、 改进等， 均应包含在本发明的保护范围之内。 The results of RT-PCR are shown in Figure 8. Compared with PPC, ICC's nestin gene and ABCG2 gene expression are weakened or disappeared, while islet-associated genes begin to express, insulin, glucagon, somatostatin and PDX-1, GLUT2. Isogenic expression was positive. Example 7 Determination of Insulin and C-peptide Secretion by ICC of Normal Islet and Induced Differentiation Under the action of different stimuli, 200 normal limbs or ICC from Example 4 were separately selected, and 0.5 ml of small centrifugation was placed. In the tube, wash twice with sugar-free DMEM for 5 minutes each time, then use 200 ul of DMEM containing different concentrations of glucose (OmM, 5.6 mM or 16.7 mM) or 200 ul of lOmML-arginine and different concentrations of glucose (OMM, respectively). 5.6 mM or 16.7 mM DMEM, incubation at 37 ° C (0 2 /C0 2 , 95:5) for 90 minutes, then collect the supernatant with hypersensitive insulin ELISA and ultrasensitive C-Peptide aspartic acid ELISA reagent The box (Mercodia, Uppsala, Sweden) was analyzed and the results are shown in Figure 9. Figure 9 shows that insulin is stimulated by ICC and adult islets at different concentrations of glucose or with 1 OmM L-arginine (Figures 9A1, 9A2) The secretion of C-peptide (Fig. 9B1, 9B2) was as follows: Under the stimulation of different concentrations of glucose, the amount of insulin secretion was (mIUx lO- 6 /min/ICC or islet): under glucose stimulation of OmM concentration: normal Insulin secretion of adult islets was 0.2174±0.0632, ICC was 0.0561±0.0258; under glucose stimulation of 5.6 mM: insulin secretion of adult islets was 0.9305±0.0896, ICC was 0.0845±0.0339; glucose stimulation at 16.7 mM the: amount of insulin secreted into body down islands is 2.0907 ± 0.2644, ICC 0.1 XIE ± 0.0542; secretion C- peptide is (pmolx l0- 6 / min / islet or ICC): at OmM concentrations of glucose stimulation: adult The C-peptide secretion of islets was 4.441±1.218, ICC was 0.0585±0.1013; under the stimulation of 5.6 mM glucose: the secretion of C-peptide of adult islets was 7.3336±1.9997, ICC was 0.1295±0.1323; at 16.7mM concentration Under the stimulation of glucose: the secretion of C-peptide in adult islets was 9.4841±3.3433, and the ICC was 0.5423±0.1382. When stimulated by different concentrations of glucose and 10mML4 lysine, the amount of insulin secreted was (mlUx lO- 6 / Min/ ICC or islet): In the simultaneous stimulation of OmM glucose and 10mML-arginine, the insulin secretion of adult islets was 0.6003±0·0419, ICC was 0.0641±0.0279; at the same time 5.6mM glucose and 10mML-arginine Under stimulation, the insulin secretion of adult islets was 2.7675±0.8867, ICC was 0.1809±0.0211. Under the simultaneous stimulation of 16.7mM glucose and lOmML-arginine, the insulin secretion of adult islets was 6.1731±2.4574, ICC was 0.2677± 0.0792; C-peptide secretion was (pmolx l0" 6 /min/islet or ICC). Under the simultaneous stimulation of OmM glucose and 10mML4 lysine, the C-peptide secretion of adult islets was 5.5077±1.2808, ICC was 0.2984. ±0.0301, stimulated by 5.6 mM glucose and lOmML-arginine, the C-peptide secretion of adult islets was 7.9897±0.4765, ICC was 0.4901±0.1382, and stimulation was performed at 16.7 mM glucose and lOmML-arginine. The C-peptide secretion of adult islets was 12.8138±2.8141, and the ICC was 0·6295±0·2471. The results show that, according to the method of the present invention, pancreatic endocrine cells of ICC structure obtainable from pancreatic cells of diabetic animals, the pancreatic endocrine cells can be To secrete insulin and C peptide. The in vitro cultured pancreatic endocrine cells can be transplanted to diabetic animals in the same or allogeneic manner, so that diabetic animals can get rid of the dependence of insulin injection. It also solves the problem of rejection of allogeneic transplantation and insufficient source of thousands of cells. In addition, the primer sequences mentioned in the present invention are artificially synthesized (which can be synthesized by Shanghai). The above is only the preferred embodiment of the present invention, and is not intended to limit the present invention, and various modifications and changes can be made to the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and scope of the present invention are intended to be included within the scope of the present invention.

Claims

权利要求书 Claim
1. 一种检测糖尿病动物胰腺组织中胰腺干细胞的方法， 包 括-. A method for detecting pancreatic stem cells in pancreatic tissue of a diabetic animal, comprising -.
a ) 免疫组化检测， 包括： 胰腺组织固定， 制作石蜡 包埋切片， 封闭后接触一抗抗体， 接触二抗抗体， 然后 于 片观察， 其中所述一抗抗体包括兔抗巢蛋白抗体；  a) immunohistochemical detection, including: pancreatic tissue fixation, preparation of paraffin-embedded sections, blocking exposure to primary antibody, exposure to secondary antibody, and then observation, wherein the primary antibody comprises a rabbit anti-nestin antibody;
b ) RT-PCR分析， 包括提取总 RNA, 通过逆转录形 成 cDNA, 然后利用引物进行 PCR扩增； c )根据步骤 a )和 b ) 的结果确定糖尿病动物夷腺组 织中是否存在胰腺干细胞。  b) RT-PCR analysis, including extraction of total RNA, formation of cDNA by reverse transcription, followed by PCR amplification using primers; c) Determination of the presence of pancreatic stem cells in the tissue of diabetic animals according to the results of steps a) and b).
2. 根据权利要求 1所述的方法， 其中步驟 b ) 中 PCR扩增使 用的引物包括巢蛋白基因的上游引物： AGAGGGGAATTC 或者上游引物 AAGAGCTGGAGGGCGTGGTG 和下游引 物 CTG TCCTGATAGCCGCGCACTG ( 328bp ) 。 2. The method according to claim 1, wherein the primer for PCR amplification in step b) comprises an upstream primer of the nestin gene: AGAGGGGAATTC or the upstream primer AAGAGCTGGAGGGCGTGGTG and the downstream primer CTG TCCTGATAGCCGCGCACTG (328bp).
3. 一种从糖尿病动物胰腺组织中分离残存的胰岛细胞团的 方法， 包括以下步骤： a ) 将胰腺组织剪碎成块； b ) 对块状的胰腺组织进行酶消化， 然后终止消化； c ) 离心分离， 去上清， 清洗； d ) 得到分离后的胰岛细胞团。 3. A method for isolating residual islet cell mass from pancreatic tissue of a diabetic animal, comprising the steps of: a) cutting the pancreatic tissue into pieces; b) enzymatically digesting the massive pancreatic tissue, and then terminating digestion; Centrifugal separation, de-clearing, washing; d) Obtaining isolated islet cell clusters.
4. 根据权利要求 3所述的方法， 其中步驟 b ) 中酶消化是在 V型胶原酶中在 37°C消化 5-10分钟。 4. The method according to claim 3, wherein the enzymatic digestion in step b) is carried out in a type V collagenase at 37 ° C for 5-10 minutes.
5. 根据权利要求 4所述的方法， 其中步骤 b ) 中通过加入等 体积的冰冷的 Hank's液终止消化。 5. A method according to claim 4, wherein in step b) the digestion is terminated by the addition of an equal volume of ice-cold Hank's solution.
6. —种胰岛干细胞的分离、 培养的方法， 包括以下步骤： a ) 将从糖尿病动物胰腺组织中分离出的胰岛细胞团 加入装有培养基 A的培养 i中 , 培养 2-5天； 6. A method for isolating and culturing islet stem cells, comprising the steps of: a) adding islet cell clusters isolated from pancreatic tissue of a diabetic animal to culture i containing medium A for 2-5 days;
b ) 将悬浮的胰岛细胞团接种于含有培养基 B的培养 b) inoculation of suspended islet cell pellets in culture containing medium B
JD1中培养； c ) 经过 6-20天， 生长出星形胰岛干细月包； d ) 在所述星形胰岛干细胞将汇合时传代， 直到长满 传代后的培养 jnr, 得到扩增的胰岛干细胞。 CD1 culture; c) After 6-20 days, the star islet dry fine moon pack is grown; d) when the star islet stem cells will be confluent, until the jnr is cultured after the passage, the amplified islets are obtained. stem cell.
7. 根据权利要求 6所述的方法， 其中， 所述培养基 A包括： 基础培养基、 緩冲液、 血清、 氨基酸和抗生素。 7. The method according to claim 6, wherein the medium A comprises: a basal medium, a buffer, serum, an amino acid, and an antibiotic.
8. 根据权利要求 7所述的方法， 其中， 所述基础培养基包括 RPMI 1640; 所述血清包括 FBS; 所述緩冲液包括 4-羟乙 基哌漆乙横緩冲液、 丙酮酸钠緩冲液； 所述氨基酸包括 L-谷氨酸； 所述抗生素包括青霉素-链霉素。 8. The method according to claim 7, wherein the basal medium comprises RPMI 1640; the serum comprises FBS; the buffer comprises 4-hydroxyethyl pipetting agent B transverse buffer, sodium pyruvate a buffer; the amino acid comprises L-glutamic acid; and the antibiotic comprises penicillin-streptomycin.
9. 根据权利要求 8所述的方法， 其中所述培养基 A包括： RPMI 1640、 8-15% FBS、 10mM 4-羟乙基派秦乙石黄緩冲液、 ImM丙酮酸钠緩冲液、 2mM L-谷氨酸、 100u青霉素 - lOOug 链霉素。 9. The method according to claim 8, wherein the medium A comprises: RPMI 1640, 8-15% FBS, 10 mM 4-hydroxyethylpyrazine buffer, 1 mM sodium pyruvate buffer, 2 mM L- Glutamate, 100u penicillin - lOOug streptomycin.
10. 根据权利要求 6所述的方法，其中， 所述培养基 A的按体 积计算的配方可以为： 86% RPMI 1640、 10%FBS、 1%HEPES ( 1M ) 、 1%丙酮酸钠 （lOOmM ) 、 1% L-谷氨 酰胺（200mM ) 、 l%10000u青霉素 - lOOOOug链霉素； 或 者 81%RPMI 1640、 15%FBS、 1%HEPES ( 1M) 、 1%丙 酮酸钠 ( 1 OOmM )、 1 % L-谷氨酰胺（ 200mM )、 1 % 1 OOOOu 青霉素 -lOOOOug链霉素； 或者 88%RPMI 1640、 8%FBS、 1%HEPES ( 1M) 、 1%丙酮酸钠 ( lOOmM) 、 1%L-谷氨 酰胺（200mM) 、 l%10000u青霉素 - lOOOOug链霉素； 或 者 43mlRPMI 1640、 5mlFBS、 0.5ml HEPES ( 100X) 、 0.5ml 丙酮酸钠 ( 100X) 、 0.5ml L-glutamine ( 100X) 、 0.5ml Antibiotic ( 100X) 。 10. The method according to claim 6, wherein the volumetric formula of the medium A can be: 86% RPMI 1640, 10% FBS, 1% HEPES (1M), 1% sodium pyruvate (100 mM) ), 1% L-glutamine (200 mM), 1% 10000 u penicillin - lOOOOug streptomycin; or 81% RPMI 1640, 15% FBS, 1% HEPES (1M), 1% sodium pyruvate (100 mM), 1% L-glutamine (200 mM), 1% 1 OOOOu penicillin-lOOOOug streptomycin; 88% RPMI 1640, 8% FBS, 1% HEPES (1M), 1% sodium pyruvate (100 mM), 1% L-glutamine (200 mM), 1% 10000 u penicillin-lOOOOug streptomycin; or 43 ml RPMI 1640, 5ml FBS, 0.5ml HEPES (100X), 0.5ml sodium pyruvate (100X), 0.5ml L-glutamine (100X), 0.5ml Antibiotic (100X).
11. 根据权利要求 6所述的方法， 其中所述培养基 B包括： 基 础培养基、 緩冲液、 血清、 氨基酸、 抗生素和生长因子。 The method according to claim 6, wherein the medium B comprises: a basic medium, a buffer, a serum, an amino acid, an antibiotic, and a growth factor.
12. 根据权利要求 11 所述的方法， 其中所述基础培养基包括 RPMI 1640; 所述血清包括 FBS; 所述緩冲液包括 4-羟乙 基哌嗪乙横緩冲液、 丙酮酸钠緩冲液； 所述氨基酸包括 L-谷氨酸； 所述抗生素包括青霉素-链霉素； 所述生长因 子包括碱性成纤维细胞生长因子、 表皮细胞生长因子。 12. The method according to claim 11, wherein the basal medium comprises RPMI 1640; the serum comprises FBS; the buffer comprises 4-hydroxyethyl piperazine B transverse buffer, sodium pyruvate buffer The amino acid includes L-glutamic acid; the antibiotic includes penicillin-streptomycin; and the growth factor includes basic fibroblast growth factor, epidermal growth factor.
13. 根据权利要求 12所述的方法， 其中所述培养基 B包括： RPMI 1640、 8-15% FBS、 10mM 4-羟乙基哌，秦乙黄緩冲液、 ImM丙酮酸钠緩冲液、 2mML-谷氨酸、 100u青霉素 -100u 链霉素、 10-20ng/ml碱性成纤维细胞生长因子、 10-20ng/ml 表皮细胞生长因子。 13. The method according to claim 12, wherein the medium B comprises: RPMI 1640, 8-15% FBS, 10 mM 4-hydroxyethylpiper, Qinhuanghuang buffer, 1 mM sodium pyruvate buffer, 2 mML - glutamic acid, 100 u penicillin-100 u streptomycin, 10-20 ng/ml basic fibroblast growth factor, 10-20 ng/ml epidermal growth factor.
14. 根据权利要求 6所述的方法，其中所述培养基 B包括： 86% RPMI 1640、 10%FBS、 1%HEPES ( 1M ) 、 1%丙酮酸钠14. The method of claim 6, wherein the medium B comprises: 86% RPMI 1640, 10% FBS, 1% HEPES (1M), 1% sodium pyruvate
( lOOmM) 、 1%L-谷義酰胺 ( 200mM ) 、 1%1 OOOOu 青 霉素- lOOOOug链霉素、 20ng/ml bFGF、 20ng/ml EGF; 或 者 81% RPMI 1640、 15% FBS, 1% HEPES ( 1M) 、 1% 丙酮酸钠（ lOOmM )、 1% L-谷氨酰胺（ 200mM )、 1%1 OOOOu 青霉素 -lOOOOug链霉素、 20ng/ml bFGF、 20ng/ml EGF; 或者 88% RPMI 1640、 8% FBS、 1% HEPES ( 1M ) 、 1% 丙酮 炱钠（ lOOmM )、 1% L-谷氨酰胺（ 200mM )、 l%10000u 青霉素 -lOOOOug链霉素、 20ng/ml bFGF, 20ng/ml EGF; 或者 43ml RPMI 1640、 5ml FBS、 0.5ml HEPES ( 100X ) 、 0.5ml 丙酮酉炱 3 ( 100X ) 、 0.5ml L-glutamine ( 100X ) 、 0.5ml Antibiotic ( 100X ) 、 20ng/ml Human bFGF, 20ng/ml Human EGF。 (100 mM), 1% L-glutamine (200 mM), 1% 1 OOOOu penicillin- lOOOOug streptomycin, 20 ng/ml bFGF, 20 ng/ml EGF; or 81% RPMI 1640, 15% FBS, 1% HEPES ( 1M), 1% sodium pyruvate (100 mM), 1% L-glutamine (200 mM), 1% 1 OOOOu Penicillin-lOOOOug streptomycin, 20 ng/ml bFGF, 20 ng/ml EGF; or 88% RPMI 1640, 8% FBS, 1% HEPES (1M), 1% sodium valproate (100 mM), 1% L-glutamine (200 mM), l% 10000 u penicillin-lOOOOug streptomycin, 20 ng/ml bFGF, 20 ng/ml EGF; or 43 ml RPMI 1640, 5 ml FBS, 0.5 ml HEPES (100X), 0.5 ml acetone oxime 3 (100X), 0.5 Ml L-glutamine (100X), 0.5ml Antibiotic (100X), 20ng/ml Human bFGF, 20ng/ml Human EGF.
15. 一种将胰岛干细胞培养成为胰腺内分泌细胞的方法， 包括 以下步一骤： a ) 夺胰岛干细胞接种于含有培养基 C的培养皿中， 培养 2-5天； A method for culturing islet stem cells into pancreatic endocrine cells, comprising the following steps: a) islet stem cells are seeded in a culture dish containing medium C, and cultured for 2-5 days;
b )将培养基 C更换为培养基 D,但仍然在培养 中， 培养大约 4-15天， 得到可分泌胰岛素的胰腺内分泌细胞。  b) Replace medium C with medium D, but still in culture, culture for about 4-15 days to obtain pancreatic endocrine cells that can secrete insulin.
16. 根据权利要求 15所述的将胰腺干细胞培养成为胰腺内分 泌细 包的方法， 其中所述培养孤为细菌培养 i或涂有 0.01%多聚鸟氛酸或多聚赖氨酸的细胞培养 。 16. The method of culturing pancreatic stem cells into a pancreatic endocrine packet according to claim 15, wherein the culture is cultured in a bacterial culture or cultured with 0.01% poly-ornic acid or polylysine.
17. 根据权利要求 15所述的方法， 其中所述培养基 C包括： 基础培养基、 培养添加剂、 白蛋白、 抗生素和生长因子。 17. The method according to claim 15, wherein the medium C comprises: a basal medium, a culture additive, albumin, an antibiotic, and a growth factor.
18. 根据权利要求 17所述的方法， 其中所述基础培养基包括 DMEM和 Nutrient Mixture Ham's F-12'; 所述培养添力口 剂包括 B27或者 lg/LITS; 所述白蛋白包括 BSA; 所述抗 生素包括青霉素-链霉素； 所述生长因子包括碱性成纤维 细胞生长因子、 表皮细胞生长因子。 18. The method according to claim 17, wherein the basal medium comprises DMEM and Nutrient Mixture Ham's F-12'; the culture addition agent comprises B27 or lg/LITS; the albumin comprises BSA; The antibiotics include penicillin-streptomycin; the growth factors include basic fibroblast growth factor, epidermal growth factor.
19. 根据权利要求 18所述的方法， 其中所述培养基 C包括： 49%DMEM、 49%Nutrient Mixture Ham's F-12，、 2%B27 或者 lg/LITS、 0.05%-0.2% BSA, lOOu青霉素 -lOOug链 霉素、 10- 20ng/ml碱性成纤维细胞生长因子、 10-20ng/ml 表皮细胞生长因子。 19. The method according to claim 18, wherein the medium C comprises: 49% DMEM, 49% Nutrient Mixture Ham's F-12, 2% B27 or lg/LITS, 0.05%-0.2% BSA, lOOu penicillin -lOOug streptomycin, 10-20 ng/ml basic fibroblast growth factor, 10-20 ng/ml epidermal growth factor.
20. 根据权利要求 15所述的方法， 其中所述培养基 C包括： 48%DMEM (无糖） 或 （3mmol/l 葡萄糖） 、 48% F12、 2% B27、 1% 5%BSA、 20ng/ml bFGF、 20ng/ml EGF、 l%10000u青霉素 -lOOOOug链霉素； 或者 48% DMEM (无 糖）或（3mmol/l葡萄糖）、 48% F12、 2% B27、 1% 10% BSA、 20ng/ml bFGF、 20ng/ml EGF、 l%10000u青霉素 -lOOOOug链霉素； 或者 48% DMEM (无糖 )或（ 3mmol/l 葡萄糖）、 48% F12、 2% B27、 1%20% BSA、 20ng/ml bFGF、 20ng/ml EGF、 l%10000u青霉素 -lOOOOug链霉素； 或者 49% DMEM (无糖 )或 （ 3mmol/l葡萄糖 ) 、 49% F12、 lg/L ITS , 1%5% BSA、 20ng/ml bFGF、 20ng/ml EGF, 1% lOOOOu青霉素 -lOOOOug链霉素；或者 49% DMEM (无糖） 或（3mmol/l葡萄糖）、 49% F12、 lg/L ITS, 1%10% BSA、 20ng/ml bFGF、 20ng/ml EGF、 l%10000u青霉素 -lOOOOug 链霉素； 或者 49%DMEM (无糖）或（ 3mmol/l葡萄糖）、 49% F12、 lg L ITS、 1%20% BSA^ 20ng/mlbFGF、 20ng/ml EGF、 1% lOOOOu青霉素 -lOOOOug链霉素；或者 DMEM/F12 1 :1 ( 8mM glucose ) 、 B27 ( 50X ) 、 0.075% BSA、 20ng/ml bFGF、 20ng/ml EGF、 Antibiotics ( 100X ) 。 20. The method according to claim 15, wherein the medium C comprises: 48% DMEM (no sugar) or (3 mmol/l glucose), 48% F12, 2% B27, 1% 5% BSA, 20 ng/ Ml bFGF, 20 ng/ml EGF, 1% 10000u penicillin-lOOOOug streptomycin; or 48% DMEM (no sugar) or (3mmol/l glucose), 48% F12, 2% B27, 1% 10% BSA, 20ng/ Ml bFGF, 20 ng/ml EGF, 1% 10000u penicillin-lOOOOug streptomycin; or 48% DMEM (no sugar) or (3mmol/l glucose), 48% F12, 2% B27, 1% 20% BSA, 20ng/ Ml bFGF, 20 ng/ml EGF, 1% 10000u penicillin-lOOOOug streptomycin; or 49% DMEM (no sugar) or (3mmol/l glucose), 49% F12, lg/L ITS, 1% 5% BSA, 20ng /ml bFGF, 20ng/ml EGF, 1% lOOOOu penicillin-lOOOOug streptomycin; or 49% DMEM (no sugar) or (3mmol/l glucose), 49% F12, lg/L ITS, 1% 10% BSA, 20ng/ml bFGF, 20ng/ml EGF, l%10000u penicillin-lOOOOug streptomycin; or 49% DMEM (no sugar) or (3mmol/l glucose), 49% F12, lg L ITS, 1% 20% BSA^ 20ng/mlbFGF, 20ng/ml EGF, 1% lOOOOu -LOOOOug streptomycin, neomycin; or DMEM / F12 1: 1 (8mM glucose), B27 (50X), 0.075% BSA, 20ng / ml bFGF, 20ng / ml EGF, Antibiotics (100X).
21. 根据权利要求 15所述的方法， 其中所述培养基 D包括： 基础培养基、 培养添加剂、 白蛋白、 抗生素、 有诱导功能 的多肽和生长因子。 21. The method of claim 15, wherein the medium D comprises: a basal medium, a culture additive, an albumin, an antibiotic, an inducible polypeptide, and a growth factor.
22. 根据杈利要求 21所述的方法， 其中所述基础培养基包括 DMEM和 Nutrient Mixture Ham's F-12';所述培养添力口剂 包括 B27或者 lg/L ITS; 所述白蛋白包括 BSA; 所述抗 生素包括青霉素-链霉素； 所述多肽和生长因子包括： 烟 酰胺、 GLP- HGF、 Betacellulin。 22. The method according to claim 21, wherein the basal medium comprises DMEM and Nutrient Mixture Ham's F-12'; the culture addition agent comprises B27 or lg/L ITS; the albumin comprises BSA The antibiotic comprises penicillin-streptomycin; the polypeptide and growth factors include: nicotinamide, GLP-HGF, Betacellulin.
23. 根据权利要求 22所述的方法， 其中所述培养基 D包括： 49%DMEM、 49%Nutrient Mixture Ham's F-12\ 2%B27 或者 lg/LITS、 0.05%-0.2% BSA, lOOu青霉素 -lOOug链 霉素、 lOmM烟酰胺、 10-lOOnM GLP-1、 lOng/ml HGF, 500pmol/L Betacelluliri。 23. The method according to claim 22, wherein the medium D comprises: 49% DMEM, 49% Nutrient Mixture Ham's F-12\2% B27 or lg/LITS, 0.05%-0.2% BSA, lOOu penicillin- lOOug streptomycin, lOmM nicotinamide, 10-lOOnM GLP-1, lOng/ml HGF, 500 pmol/L Betacelluliri.
24. 根据权利要求 15所述的方法， 其中所述培养基 D包括： 48%DMEM (无糖 )或 ( 3mmol/l葡萄糖）、 48%F12、 2% B27 1% 的 5% BSA、 10mM烟酰胺、 10nM GLP-1 ( 7-36 amide ) 、 lOng/ml HGF、 500pM Betacellulin, l%10000u 青霉素 -lOOOOug 链霉素； 或者 48%DMEM (无糖） 或24. The method according to claim 15, wherein the medium D comprises: 48% DMEM (no sugar) or (3 mmol/l glucose), 48% F12, 2% B27 1% 5% BSA, 10 mM smoke Amide, 10nM GLP-1 (7-36 amide), lOng/ml HGF, 500pM Betacellulin, l%10000u penicillin-lOOOOug streptomycin; or 48% DMEM (no sugar) or
( 3mmol/l葡萄糖 )、 48% F12、 2% B27、 1%的 10% BSA、 lOmM 因酰胺、 10nM GLP-1 ( 7-36 amide )、 lOng/ml HGF、 500pM Betacellulin, l%10000u青霉素 - lOOOOug链霉素； 或者 48% DMEM(无糖）或（3mmol/l葡萄糖）、 48% F12、 2% B27、 1%20% BSA、 lOmM 因醜胺、 10nM GLP-1 ( 7-36 amide ) 、 lOng/ml HGF、 500pM Betacellulin、 l%10000u 青霉素 -lOOOOug 链霉素； 或者 49% DMEM (无糖） 或(3mmol/l glucose), 48% F12, 2% B27, 1% 10% BSA, lOmM amide, 10nM GLP-1 (7-36 amide), lOng/ml HGF, 500pM Betacellulin, 1% 10000u penicillin- lOOOOug streptomycin; or 48% DMEM (no sugar) or (3mmol/l glucose), 48% F12, 2% B27, 1% 20% BSA, lOmM ugly amine, 10nM GLP-1 (7-36 amide) , lOng/ml HGF, 500pM Betacellulin, l%10000u penicillin-lOOOOug streptomycin; or 49% DMEM (no sugar) or
( 3mmol/l 葡萄糖） 、 49% F12、 lg/L ITS > 1%5% BSA、 lOmM 因酰胺、 ΙΟηΜ GLP-1 ( 7-36 amide )、 lOng/ml HGF、 500pM Betacellulin> l%10000u青霉素 -lOOOOug链霉素； 49% DMEM (无糖）或 （ 3mmol/l葡萄糖） 、 49% F12、 lg/L ITS, 1%10%BSA、 lOmM烟酰胺、 ΙΟηΜ GLP-1 ( 7-36 amide ) 、 lOng/ml HGF、 500pM Betacellulin, l%10000u 青霉素 -lOOOOug链霉素； 49% DMEM (无糖 )或（ 3mmol/l 葡萄糖） 、 49% F12、 lg/L ITS, 1%20% BSA、 10mM烟 酰胺、 lOnM GLP-1 ( 7-36 amide ) 、 10ng/ml HGF、 500pM Betacellulin, l%10000u 青霉素 -lOOOOug 链霉素； 或者 DMEM/F12 1 : 1 ( 5.6 Mm glucose ) 、 B27 ( 50X ) 、 0.075% BSA、 lOmM nicotinamide > Antibiotics( 100X )、 ΙΟηΜ GLP-1 ( 7-36 amide ) 、 lOng/ml HGF, 500pmol/L Betacellulin。 (3mmol/l glucose), 49% F12, lg/L ITS > 1% 5% BSA, lOmM amide, ΙΟηΜ GLP-1 (7-36 amide), lOng/ml HGF, 500pM Betacellulin> l%10000u penicillin- lOOOOug streptomycin; 49% DMEM (no sugar) or (3mmol/l glucose), 49% F12, lg/L ITS, 1% 10% BSA, lOmM nicotinamide, ΙΟηΜ GLP-1 (7-36 amide ), lOng/ml HGF, 500pM Betacellulin, l%10000u Penicillin-lOOOOug streptomycin; 49% DMEM (no sugar) or (3mmol/l glucose), 49% F12, lg/L ITS, 1% 20% BSA, 10 mM nicotinamide, lOnM GLP-1 (7-36 amide ), 10 ng/ml HGF, 500 pM Betacellulin, l% 10000 u penicillin-lOOOOug streptomycin; or DMEM/F12 1 : 1 (5.6 Mm glucose ), B27 (50X), 0.075% BSA, lOmM nicotinamide > Antibiotics (100X), ΙΟηΜ GLP-1 (7-36 amide ), lOng/ml HGF, 500 pmol/L Betacellulin.
25. 利用权利要求 3至 5中任一项所述的从糖尿病动物胰腺组 织中分离得到的腹岛细月包团。 25. The ascites granules isolated from the pancreas tissue of a diabetic animal according to any one of claims 3 to 5.
26. 利用权利要求 6至 14中任一项所述的胰岛干细胞的分离、 培养的方法得到的胰岛干细胞， 所述肢岛干细胞为多角形 细胞， 其中巢蛋白基因表达为阳性。 利用权利要求 15至 24中任一项所述的将胰岛干细胞培养 成为胰腺内分泌细胞的方法得到的胰腺内分泌细胞。 The islet stem cells obtained by the method for isolating and culturing the islet stem cells according to any one of claims 6 to 14, wherein the limbic stem cells are polygonal cells, wherein the nestin gene expression is positive. A pancreatic endocrine cell obtained by the method of culturing islet stem cells into pancreatic endocrine cells according to any one of claims 15 to 24.
PCT/CN2006/001669 2005-07-15 2006-07-14 A method for detecting and culturing pancreatic cells and their application WO2007009356A1 (en)

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