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WO2007009356A1 - Méthode pour détecter et cultiver des cellules pancréatiques et application - Google Patents

Méthode pour détecter et cultiver des cellules pancréatiques et 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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Chinese (zh)
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Yu Zhang
Chunlin Zou
Biao Chen
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THERACELLS BIOTECHNOLOGIES CO Ltd
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THERACELLS BIOTECHNOLOGIES CO Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C—CHEMISTRY; METALLURGY
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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
    • 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
    • G01N33/507—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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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
    • C12N2500/24—Iron; Fe chelators; Transferrin
    • C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • C—CHEMISTRY; METALLURGY
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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

L’invention concerne des méthodes d'isolement et de culture de cellules souche pancréatiques et de différencier des cellules souche pancréatiques en cellules endocrines pancréatiques qui sécrètent de l'insuline. Ces méthodes comprennent : l'isolation de masse cellulaire d’îlots pancréatiques provenant du pancréas d'animaux atteints du diabète ; la culture de la masse cellulaire isolée en milieu A pendant 2 à 5 jours ; la culture de la masse cellulaire isolée en milieu B pendant 6 à 20 jours pour produire des cellules souche d'îlots pancréatiques en étoile ; la sous-culture des cellules souche d'îlots pancréatiques en étoile pour produire des cellules souche d'îlots pancréatiques. Ces cellules souche d'îlots pancréatiques sont inoculées en milieu C et sont cultivées pendant 4 à 15 jours. Ensuite le milieu D est substitué au milieu C, et les cellules souche d'îlots pancréatiques sont cultivées pendant 4 à 15 jours supplémentaires pour produire des cellules endocrines pancréatiques qui secrète de l'insuline.
PCT/CN2006/001669 2005-07-15 2006-07-14 Méthode pour détecter et cultiver des cellules pancréatiques et application Ceased WO2007009356A1 (fr)

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US12369585B2 (en) 2017-03-07 2025-07-29 Upl Ltd Fungicidal combinations
US12376592B2 (en) 2017-03-07 2025-08-05 Upl Ltd Fungicidal combinations
CN113481147A (zh) * 2021-06-10 2021-10-08 艾可泰科生物科技(江苏)有限公司 人胰岛来源的胰腺干细胞系构建及向胰岛细胞分化的方法

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