小鼠胰腺导管上皮细胞和骨髓间充质干细胞转分化为胰岛样细胞的实验研究
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摘要
目 的:
1 型糖尿病的发生是由于胰岛素的绝对缺乏,需终身依赖胰岛素治疗,但是胰岛素治疗并不能完全阻止糖尿病并发症的发生,因此寻找合适的细胞以替代β细胞发挥生理功能具有重要的意义。Shapiro 等报道,给 7 例 1 型糖尿病患者移植新鲜胰岛并配合非糖皮质激素免疫抑制剂治疗,患者不依赖外源胰岛素而血糖控制良好的平均时间超过了 12 个月,可是,供体来源的匮乏以及移植免疫排斥反应极大地阻碍了该方法的推广和应用。体外培养干细胞使其定向分化为胰岛样细胞以替代患者体内受损胰岛的生理功能,可能是解决供体来源匮乏以及移植免疫排斥反应有效方法之一。研究证实小鼠胰腺导管上皮细胞能够分化为具有内分泌功能的胰岛样细胞, 本研究采用细胞培养及诱导分化技术,分离培养小鼠胰腺导管上皮细胞并使之转分化为胰腺干细胞及胰岛素分泌细胞(胰岛样细胞),并将胰岛样细胞移植入 1 型糖尿病小鼠体内,定期观察血糖水平 2 个月,为胰岛细胞移植治疗糖尿病奠定实践基础。
材料与方法:
1、 小鼠胰腺导管上皮细胞原代培养、诱导分化及鉴定:实验用成年昆明小鼠,以Ⅴ型胶原酶消化加滤网过滤法,分离小鼠胰腺导管上皮细胞,用添加有多种生长因子的 DMEM/F12 培养基培养并诱导分
Objectives
Patients with type 1 diabetes must be treated with insulin throughout life, and the treatment can’t prevent complications completely .So it is very important to find a new method replacingβ-cell function in vivo. A recent report by Shapiro has demonstrated that using a glucocorticoid-free immunosuppressive therapy combined with the infusion of an adequate fresh islet mass resulted in insulin independence and good metabolic control for periods of more than 12 months in 7 patients with type 1 diabetes. However, such an approach is limited by the scarcity of the transplant and the long-term side effects of immunosuppressive therapy. These problems may be overcome by using a renewable source of cells, such as islet-cells derived from stem cells, which provide us a new strategy to treatment of diabetes: transdifferentiate stem cells into islet cells in vitro in order to replace disfunctioning βcells of diabetic patients. There is clear evidence that stem cells committed to differentiate into insulin-secreting cells, serve as a possible future source for cell- replacement therapy in diabetes. In order to look for suitable cell-replacement source and improve the technique of stem cells, pancreatic ductal epithelial cells from Kunming mice were separated, cultured, transdifferentiated into insulin-producing cells and tansplanted into abdominal cavity of mice in the experiment. This led us to investigate
whether differentiated islet-like clusters could be stimulated to produce insulin in vivo.
Materials and Methods
Pancreas of Kunming mice were digested with collagenase type Ⅴ, followed by filtrating to separate pancreatic ductal epithelial cells from islets and acinar tissue. Ductal epithelial cells were cultivated in DMEM/F12 medium with the addition of growth factors. Samples were taken at different time points for light and electronic microscopic examination and for immunocytochemical study with antibodies against transdifferentiation gene PDX-1 and protein CK-19. The expression of insulin and glucagon genes was determined by RT-PCR. DTZ stain experiments were done to examine whether differentiated islet-like clusters had the function of producing insulin. In order to investigate whether differentiated islet-like clusters could be stimulated to differentiate into functional islets if placed in vivo environment, islet-like clusters transplantation was performed. Model mice of type 1diabetes made by streptozotocin injection into abdominal cavity must have high blood glucose levels over 16.6mmol/L twice a days, The diabetic mice were divided into two groups (n=6) ad libitum, 6 diabetic mice in each group, and there were 6 normal mice as control (n=6). Group 1 was experimental mice implanted with islets about 3500 equivalent into abdominal cavity, group 2 was implanted same volume of normal saline. Group 3 was normal control implanted same volume of normal saline. All mice were examined for blood glucose levels and observed for life conditions regularly. Experiment period was 2 months.
Results
1. Primary culture of pancreatic ductal epithelial cells from Kunming
mice: At the beginning of separation, typical epithelioid cells existed in single globular shape. These cells proliferated quickly when the medium had been changed to full-medium 48 hours later. These cells then grew for about 1-2 weeks until reaching near confluence or forming substantial plaques of epithelial cells in cobblestone pattern, epithelioid cells gathered gradually and formed islet-like clusters 2-3 weeks later.
2. Immunocytochemistry: At the beginning of isolation a large number of epithelioid cells were CK-19 immunoreactive positive and few of them were PDX-1 positive, while the number of CK-19+ cells increased significantly and most cells were PDX-1+ on the 16th day. It proved that pancreatic ductal epithelial cells could proliferate quickly by this way and had the potency of transdifferentiation into pancreatic stem cells.
3. RT-PCR inspecting the expression of insulin and glucagon: The analysis of mRNA by RT-PCR showed ve
1 型糖尿病的发生是由于胰岛素的绝对缺乏,需终身依赖胰岛素治疗,但是胰岛素治疗并不能完全阻止糖尿病并发症的发生,因此寻找合适的细胞以替代β细胞发挥生理功能具有重要的意义。Shapiro 等报道,给 7 例 1 型糖尿病患者移植新鲜胰岛并配合非糖皮质激素免疫抑制剂治疗,患者不依赖外源胰岛素而血糖控制良好的平均时间超过了 12 个月,可是,供体来源的匮乏以及移植免疫排斥反应极大地阻碍了该方法的推广和应用。体外培养干细胞使其定向分化为胰岛样细胞以替代患者体内受损胰岛的生理功能,可能是解决供体来源匮乏以及移植免疫排斥反应有效方法之一。研究证实小鼠胰腺导管上皮细胞能够分化为具有内分泌功能的胰岛样细胞, 本研究采用细胞培养及诱导分化技术,分离培养小鼠胰腺导管上皮细胞并使之转分化为胰腺干细胞及胰岛素分泌细胞(胰岛样细胞),并将胰岛样细胞移植入 1 型糖尿病小鼠体内,定期观察血糖水平 2 个月,为胰岛细胞移植治疗糖尿病奠定实践基础。
材料与方法:
1、 小鼠胰腺导管上皮细胞原代培养、诱导分化及鉴定:实验用成年昆明小鼠,以Ⅴ型胶原酶消化加滤网过滤法,分离小鼠胰腺导管上皮细胞,用添加有多种生长因子的 DMEM/F12 培养基培养并诱导分
Objectives
Patients with type 1 diabetes must be treated with insulin throughout life, and the treatment can’t prevent complications completely .So it is very important to find a new method replacingβ-cell function in vivo. A recent report by Shapiro has demonstrated that using a glucocorticoid-free immunosuppressive therapy combined with the infusion of an adequate fresh islet mass resulted in insulin independence and good metabolic control for periods of more than 12 months in 7 patients with type 1 diabetes. However, such an approach is limited by the scarcity of the transplant and the long-term side effects of immunosuppressive therapy. These problems may be overcome by using a renewable source of cells, such as islet-cells derived from stem cells, which provide us a new strategy to treatment of diabetes: transdifferentiate stem cells into islet cells in vitro in order to replace disfunctioning βcells of diabetic patients. There is clear evidence that stem cells committed to differentiate into insulin-secreting cells, serve as a possible future source for cell- replacement therapy in diabetes. In order to look for suitable cell-replacement source and improve the technique of stem cells, pancreatic ductal epithelial cells from Kunming mice were separated, cultured, transdifferentiated into insulin-producing cells and tansplanted into abdominal cavity of mice in the experiment. This led us to investigate
whether differentiated islet-like clusters could be stimulated to produce insulin in vivo.
Materials and Methods
Pancreas of Kunming mice were digested with collagenase type Ⅴ, followed by filtrating to separate pancreatic ductal epithelial cells from islets and acinar tissue. Ductal epithelial cells were cultivated in DMEM/F12 medium with the addition of growth factors. Samples were taken at different time points for light and electronic microscopic examination and for immunocytochemical study with antibodies against transdifferentiation gene PDX-1 and protein CK-19. The expression of insulin and glucagon genes was determined by RT-PCR. DTZ stain experiments were done to examine whether differentiated islet-like clusters had the function of producing insulin. In order to investigate whether differentiated islet-like clusters could be stimulated to differentiate into functional islets if placed in vivo environment, islet-like clusters transplantation was performed. Model mice of type 1diabetes made by streptozotocin injection into abdominal cavity must have high blood glucose levels over 16.6mmol/L twice a days, The diabetic mice were divided into two groups (n=6) ad libitum, 6 diabetic mice in each group, and there were 6 normal mice as control (n=6). Group 1 was experimental mice implanted with islets about 3500 equivalent into abdominal cavity, group 2 was implanted same volume of normal saline. Group 3 was normal control implanted same volume of normal saline. All mice were examined for blood glucose levels and observed for life conditions regularly. Experiment period was 2 months.
Results
1. Primary culture of pancreatic ductal epithelial cells from Kunming
mice: At the beginning of separation, typical epithelioid cells existed in single globular shape. These cells proliferated quickly when the medium had been changed to full-medium 48 hours later. These cells then grew for about 1-2 weeks until reaching near confluence or forming substantial plaques of epithelial cells in cobblestone pattern, epithelioid cells gathered gradually and formed islet-like clusters 2-3 weeks later.
2. Immunocytochemistry: At the beginning of isolation a large number of epithelioid cells were CK-19 immunoreactive positive and few of them were PDX-1 positive, while the number of CK-19+ cells increased significantly and most cells were PDX-1+ on the 16th day. It proved that pancreatic ductal epithelial cells could proliferate quickly by this way and had the potency of transdifferentiation into pancreatic stem cells.
3. RT-PCR inspecting the expression of insulin and glucagon: The analysis of mRNA by RT-PCR showed ve
引文
[1] Watt FM, Hogan BL. Out of Eden: stem cells and their niches[J]. Science, 2000,287(5457):1427-1430.
[2] Bretzel RG, Eckhard M, Brendel MD, et al. Pancreatic islet and stem cell transplantation: newstrategies in cell therapy of diabetes mellitus[J]. Panminerva Med. 2004 Mar; 46(1):25-42.
[3] Trucco M. Regeneration of the pancreatic beta cell[J]. J Clin Invest. 2005 Jan;115(1):5-12.
[4] Soria B,Skoudy A,Martin F, et al.From stem cells to beta cells:new strategies in cell therapy of diabetes mellitus[J].Diabetologia,2001,44(4):407-415.
[5] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen[J]. N Engl J Med,2000, 343(4):230-238.
[6] Smaglik P. Diabetes therapy boosts stem-cell campaign [J]. Nature, 2000,406 (6793):224-231.
[7] Hori H, Nakayama T, Inoue K, et al. Regenerative medicine for pancreatic beta cells[J]. Nippon Geka Gakkai Zasshi. 2004 Aug;105(8):445-453.
[8] Samstein B, Platt JL.Physiologic and immunologic hurdles to transplantation[J]. J Am Soc Nephrol,2001,12(1):182-193.
[9] Milne HM, Burns CJ, Kitsou-Mylona I, et al.Generation of insulin-expressing cells from mouse embryonic stem cells[J]. Biochem Biophys Res Commun. 2005, 328(2):399-403.
[10] Efrat S. Genetically engineered pancreatic beta-cell lines for cell therapy of diabetes[J]. Ann N Y Acad Sci, 1999,18(875):286-93.
[11] Soria B, Andreu E,Berha G, et al. Engineering pancreatic islets[J].Pflugers Arch, 2000 ,440(1):1-18.
[12] Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution [J] .Cell,2000,100(1):157-168.
[13] Nygaar D,Jensen J. Cell therapy of diabetes [J]. Adv Exp Med Biol. 2004;552:16-38.
[14] Peck AB, Ramiya V. In vitro-generation of surrogate islets from adult stem cells[J]. Transpl Immunol. 2004 Apr;12(3-4):259-272.
[15] Bouwens L, Pipeleers DG. Extra-insular beta cells associated with ductules are frequent in adult human pancreas[J]. Diabetologia,1998,41(6):629-633.
[16] Beattie G.M, Cirulli V, Lopez AD, et al. Ex vivo expansion of human pancreatic endocrine cells[J].J Clin Endocrinol Metab,1997, 82(6):1852-1856.
[17] Beattie G.M, Itkin-Ansari P, Cirulli V, et al. Sustained proliferation of PDX-1+ cells derived from human islets[J].Diabetes, 1999,48(5):1013-1019.
[18] Kerr-Conte J, Pattou F, Lecomte-Houcke M, et al. Ductal cyst formation in collagen-embedded adult human islet preparations: A means to the reproduction of nesidioblastosis in vitro[J]. Diabetes,1996,45(8):1108-1114.
[19] Yuan S, Rosenberg L, Paraskevas S,et al . Transdifferentiation of human islets to pancreatic ductal cells in collagen matrix culture[J]. Differentiation, 1996,61(1):67-75.
[20] Lefebvre VH, Otonkoski T, Ustinov J, Huotari MA, et al. Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells[J]. Diabetes,1998,47(1):134-137.
[21] Holland AM, Gonez LJ, Harrison LC, et al. Progenitor cells in the adult pancreas[J]. Diabetes Metab Res Rev. 2004 Jan-Feb;20(1):13-27.
[22] Scaglia L, Cahill CJ, FineGood DT, et al .Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal rat[J]. Endocrinology, 1997,138(4):1736-1741.
[23] Bonner-weir S, Sharma A. Pancreatic stem cells [J]. J Pathol, 2002, 197(4): 519-526.
[24] Bonner-Weir S, Taneja M, Weir GC, et al. In vitro cultivation of human islets from expanded ductal tissue [J].Proc Natl Acad Sci U.S.A, 2000,97 (14): 7999-8004.
[25] Ramiya VK, Maraist M, Arfors KE, et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells[J].Nat Med, 2000,6(3):278-282.
[26] Bonner-Weir S, Toschi E, Inada A, et al.The pancreatic ductal epithelium serves as a potential pool of progenitor cells[J]. Pediatr Diabetes. 2004;5 Suppl 2:16-22.
[27] Schmied BM, Liu G, Matsuzaki H, et al. Differentiation of islet cells in long-term culture[J]. Pancreas, 2000,20(4):337-347.
[28] Berger A.Transplanted pancreatic stem cells can reverse diabetes in mice[J]. BMJ, 2000,320(7237):736A.
[29] Leiter EH, Prochazka M,Coleman DL, et al.Animal model of human disease,the non-obese diabetic(NOD) mouse [J].Am J Path,1987,128(2):380-383.
[30] Cornelius JG,Tchernev V,Peck AB, et al.In vitro generation of islets in long-term cultures of pluripotent stem cells from adult mouse pancreas[J].Hom Metab Res,1997,29(6):271-277.
[31] Ricordi JM, Armstrong RC. In vivo proliferation of oligodendrocyte progenitors expressing PDGF alphaR during early remyelination[J]. J Neurobiol, 1998,37(3):413-428.
[32] Shewade YM, Umranim M, Bhonde RR, et al. Lage-scale isolation of islets by tissue culture of adult mouse pancreas[J].Transplant Proc, 1999,31 (3): 1721-1723.
[33] rakowski ML, Kritzik MR, Jones EM, et al.Pancreatic expression of Keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells[J].Am J Pathol,1999,154(3):683-691.
[34] 司徒镇强 吴军正. 细胞培养[M]. 世界图书出版公司,2003.1.
[35] Leibowitz G, Ferber S, Apelqvist A, et al.IPF1/PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal With type 2 diabetes [J].Diabetes,2001,50(8): 1799-1806.
[36] Otonkoski T, Beattie GM, Mally M, et al. Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells[J].J Clin Invest,1993,92(3):1459-1466.
[37] Lefebvre VH, Otonkoski T, Ustinov J, et al. Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells[J]. Diabetes, 1998,(1):134-137.
[38] Cras-Meneur C, Elghazi L,Czernichow P, et al.Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation[J].Diabetes, 2001, 50(7):1571-1579.
[39] Rooman I, Schuit F, Bouwens L, et al. Effect of vascular endothelial growth factor on growth and differentiation of pancreatic ductal epithelium [J].Lab Invest,1997,76(2):225-232.
[40] Itkin-Ansari P, Geron I, Hao E, et al.Cell-based therapies for diabetes: progress towards a transplantable human beta cell line[J]. Ann N Y Acad Sci. 2003 ,1005:138-147.
[41] Ferber S, Halkin A,Cohen H, et al. PDX-1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia[J].Nat Med,2000, 6(5): 568-572
[42] Docherty K.Growth and development of the islets of Langerhans: implications for the treatment of diabetes mellitus [J]. Curr Opin Pharmacol, 2001,1(6):641-650.
[43] Mckinnon CM, Docherty k. Pancreatic duodenal homeobox-1, PDX-1, a major regulator of beta cell identity and function[J]. Diabetologia, 2001,44 (10): 1203-1214.
[44] Guz Y, Montminy MR,Stein R, et al.Expression of murine STF-1, a putative insulin gene transcription factor, in beta cells of pancreas, duodenal epithelium and pancreatic exocrine and endocrine progenitors during ontogeny[J]. Development, 1995,121(1):11-18.
[45] Itkin-Ansari P, Levine F. Sources of beta-Cells for Human Cell-Based Therapies for Diabetes[J]. Cell Biochem Biophys. 2004;40 (3 Suppl):103-12.
[46] 周毅,许评,宋一民等.测定胰腺干细胞分子标记的临床意义[J].国外医学—消化系统分册,2001,21(3):177-180.
[47] Gmyr V, Kerr-Conte J, Belaich S, et al. Adult human cytokeratin 19-positive cells reexpress insulin promoter factor 1 in vitro: further evidence for pluripotent pancreatic stem cells in humans[J]. Diabetes,2000, 49(10) : 1671 - 1680.
[48] 杨绛,张世馥. 胰岛干细胞研究进展[J].基医学础与临床,2002,22(5): 397-403.
[49] Katdare MR, Bhonde RR, Parab PB, et al. Analysis of morphological and functional maturation of neoislets generated in vitro from pancreatic ductal cells and their suitability for islet banking and transplantation[J]. J Endocrinol. 2004 , 182(1):105-112.
[50] Kaczorowski DJ,Patterson ES,Jastromb WE, et al.G1ucose-responsive insulin-producing cells from stem cells [J].Diabetes Metab Res Rev,2002, 18(6):442-450.
[51] Lechner A, Habener JF. Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus[J]. Am J Physiol Endocrinol Metab. 2003,284(2): E259-66.
[2] Bretzel RG, Eckhard M, Brendel MD, et al. Pancreatic islet and stem cell transplantation: newstrategies in cell therapy of diabetes mellitus[J]. Panminerva Med. 2004 Mar; 46(1):25-42.
[3] Trucco M. Regeneration of the pancreatic beta cell[J]. J Clin Invest. 2005 Jan;115(1):5-12.
[4] Soria B,Skoudy A,Martin F, et al.From stem cells to beta cells:new strategies in cell therapy of diabetes mellitus[J].Diabetologia,2001,44(4):407-415.
[5] Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen[J]. N Engl J Med,2000, 343(4):230-238.
[6] Smaglik P. Diabetes therapy boosts stem-cell campaign [J]. Nature, 2000,406 (6793):224-231.
[7] Hori H, Nakayama T, Inoue K, et al. Regenerative medicine for pancreatic beta cells[J]. Nippon Geka Gakkai Zasshi. 2004 Aug;105(8):445-453.
[8] Samstein B, Platt JL.Physiologic and immunologic hurdles to transplantation[J]. J Am Soc Nephrol,2001,12(1):182-193.
[9] Milne HM, Burns CJ, Kitsou-Mylona I, et al.Generation of insulin-expressing cells from mouse embryonic stem cells[J]. Biochem Biophys Res Commun. 2005, 328(2):399-403.
[10] Efrat S. Genetically engineered pancreatic beta-cell lines for cell therapy of diabetes[J]. Ann N Y Acad Sci, 1999,18(875):286-93.
[11] Soria B, Andreu E,Berha G, et al. Engineering pancreatic islets[J].Pflugers Arch, 2000 ,440(1):1-18.
[12] Weissman IL. Stem cells: units of development, units of regeneration, and units in evolution [J] .Cell,2000,100(1):157-168.
[13] Nygaar D,Jensen J. Cell therapy of diabetes [J]. Adv Exp Med Biol. 2004;552:16-38.
[14] Peck AB, Ramiya V. In vitro-generation of surrogate islets from adult stem cells[J]. Transpl Immunol. 2004 Apr;12(3-4):259-272.
[15] Bouwens L, Pipeleers DG. Extra-insular beta cells associated with ductules are frequent in adult human pancreas[J]. Diabetologia,1998,41(6):629-633.
[16] Beattie G.M, Cirulli V, Lopez AD, et al. Ex vivo expansion of human pancreatic endocrine cells[J].J Clin Endocrinol Metab,1997, 82(6):1852-1856.
[17] Beattie G.M, Itkin-Ansari P, Cirulli V, et al. Sustained proliferation of PDX-1+ cells derived from human islets[J].Diabetes, 1999,48(5):1013-1019.
[18] Kerr-Conte J, Pattou F, Lecomte-Houcke M, et al. Ductal cyst formation in collagen-embedded adult human islet preparations: A means to the reproduction of nesidioblastosis in vitro[J]. Diabetes,1996,45(8):1108-1114.
[19] Yuan S, Rosenberg L, Paraskevas S,et al . Transdifferentiation of human islets to pancreatic ductal cells in collagen matrix culture[J]. Differentiation, 1996,61(1):67-75.
[20] Lefebvre VH, Otonkoski T, Ustinov J, Huotari MA, et al. Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells[J]. Diabetes,1998,47(1):134-137.
[21] Holland AM, Gonez LJ, Harrison LC, et al. Progenitor cells in the adult pancreas[J]. Diabetes Metab Res Rev. 2004 Jan-Feb;20(1):13-27.
[22] Scaglia L, Cahill CJ, FineGood DT, et al .Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal rat[J]. Endocrinology, 1997,138(4):1736-1741.
[23] Bonner-weir S, Sharma A. Pancreatic stem cells [J]. J Pathol, 2002, 197(4): 519-526.
[24] Bonner-Weir S, Taneja M, Weir GC, et al. In vitro cultivation of human islets from expanded ductal tissue [J].Proc Natl Acad Sci U.S.A, 2000,97 (14): 7999-8004.
[25] Ramiya VK, Maraist M, Arfors KE, et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells[J].Nat Med, 2000,6(3):278-282.
[26] Bonner-Weir S, Toschi E, Inada A, et al.The pancreatic ductal epithelium serves as a potential pool of progenitor cells[J]. Pediatr Diabetes. 2004;5 Suppl 2:16-22.
[27] Schmied BM, Liu G, Matsuzaki H, et al. Differentiation of islet cells in long-term culture[J]. Pancreas, 2000,20(4):337-347.
[28] Berger A.Transplanted pancreatic stem cells can reverse diabetes in mice[J]. BMJ, 2000,320(7237):736A.
[29] Leiter EH, Prochazka M,Coleman DL, et al.Animal model of human disease,the non-obese diabetic(NOD) mouse [J].Am J Path,1987,128(2):380-383.
[30] Cornelius JG,Tchernev V,Peck AB, et al.In vitro generation of islets in long-term cultures of pluripotent stem cells from adult mouse pancreas[J].Hom Metab Res,1997,29(6):271-277.
[31] Ricordi JM, Armstrong RC. In vivo proliferation of oligodendrocyte progenitors expressing PDGF alphaR during early remyelination[J]. J Neurobiol, 1998,37(3):413-428.
[32] Shewade YM, Umranim M, Bhonde RR, et al. Lage-scale isolation of islets by tissue culture of adult mouse pancreas[J].Transplant Proc, 1999,31 (3): 1721-1723.
[33] rakowski ML, Kritzik MR, Jones EM, et al.Pancreatic expression of Keratinocyte growth factor leads to differentiation of islet hepatocytes and proliferation of duct cells[J].Am J Pathol,1999,154(3):683-691.
[34] 司徒镇强 吴军正. 细胞培养[M]. 世界图书出版公司,2003.1.
[35] Leibowitz G, Ferber S, Apelqvist A, et al.IPF1/PDX1 deficiency and beta-cell dysfunction in Psammomys obesus, an animal With type 2 diabetes [J].Diabetes,2001,50(8): 1799-1806.
[36] Otonkoski T, Beattie GM, Mally M, et al. Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells[J].J Clin Invest,1993,92(3):1459-1466.
[37] Lefebvre VH, Otonkoski T, Ustinov J, et al. Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta-cells[J]. Diabetes, 1998,(1):134-137.
[38] Cras-Meneur C, Elghazi L,Czernichow P, et al.Epidermal growth factor increases undifferentiated pancreatic embryonic cells in vitro: a balance between proliferation and differentiation[J].Diabetes, 2001, 50(7):1571-1579.
[39] Rooman I, Schuit F, Bouwens L, et al. Effect of vascular endothelial growth factor on growth and differentiation of pancreatic ductal epithelium [J].Lab Invest,1997,76(2):225-232.
[40] Itkin-Ansari P, Geron I, Hao E, et al.Cell-based therapies for diabetes: progress towards a transplantable human beta cell line[J]. Ann N Y Acad Sci. 2003 ,1005:138-147.
[41] Ferber S, Halkin A,Cohen H, et al. PDX-1 induces expression of insulin genes in liver and ameliorates streptozotocin-induced hyperglycemia[J].Nat Med,2000, 6(5): 568-572
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