【作者】 秦贵军；

【导师】 张素华； 宋方洲；

【作者基本信息】 重庆医科大学 ， 内科学， 2005， 博士

【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更多还原

【Abstract】 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 Kunmingmice: 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更多还原