BACKGROUND: Embryonic stem cells or adult stem cells, which are both derived from human or animals, have been proved to have the ability of differentiating into insulin-producing cells, but it is necessary to evaluate their function of secreting insulin and the effect on experimental diabetes.OBJECTIVE: To evaluate the insulin secretion function in vitro by rat pancreatic stem cells after differentiation and their potential application for experimental diabetes therapy in vivo.DESIGN: Randomized control observations.SETTING: Institute of Clinical Medical Sciences in China-Japan Friendship Hospital (Beijing, China).MATERIALS: The experiment was carried out in the Laboratory of Pathophysiology, Institute of Clinical Medical Sciences,China-Japan Friendship Hospital between August 2004 and December 2007. SD male rats and female nude mice were purchased from Beijing Vital River Lab Animal Technology Co., Ltd. All the processes were obeyed by the ethical standard of animals. Nicotinamide and streptozocin were purchased from Sigma Company; anti-nestin monoclonal antibody was purchased from BD Biosciences; radio-immune-detection kit for insulin was purchased from Linco Research Company; anti-insulin antibody and anti-glucagon antibody were purchased from Santa Cruz Company,METHODS: Rat stem cells were isolated and purified from pancreatic duct cells using anti-nestin monoclonal antibody conjugated magnetic beads. These stem cells were induced to proliferate, differentiate and form insulin-producing cells in vitro. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of insulin mRNA during the cell differentiation. The immunofluorescence staining was used to detect the endocrine cells in islet-like structure induced from stem cells. The radioimmunoassay was used to detect the insulin release from stem cell differentiated islets. The native and stem cell differentiated islets were respectively transplanted under the capsule of left kidney in diabetic nude mice and glycemia was measured to evaluate the function of grafted islets.MAIN OUTCOME MEASURES: Expression of insulin mRNA during the cell differentiation. Analysis of insulin-positive cells and glucagon-positive cells in islet-like structure. Insulin release in stem cell differentiated islets. Glycemia change in diabetic mice after islet transplantation.RESULTS: RT-PCR revealed that, the expression of insulin mRNA was significantly up-regulated in stem cells during differentiation, Immunofluorescence staining indicated that there were lots of insulin-positive cells in the middle of islet-like structure and a few of glucagon-positive cells around border of the islet-like structure. Insulin analysis found that fast phase-release of insulin was absent in the stem cell differentiated islets in response to dextrose stimulation and released insulin was 39% as compared with that in native islets. Glycemia was decreased to 10 mmol/L at the 3rd day in native islet group, and then maintained at a normalized level for 60 days after transplantation; while glycemia was achieved to 10 mmol/L at the 8th day in stem cell differentiated islet group after transplantation. However, glycemia was increased gradually from the 35th day after transplantation in stem cell differentiated islet group.CONCLUSION: The stem cells derived from rat pancreas can be induced to proliferate and differentiate into insulin-producing cells and to form islet-like structure. The stem cell differentiated islet could ameliorate the glycometabolic disorder in diabetic animals.%背景:有实验证实人和动物起源的胚胎干细胞或成体干细胞可分化为胰岛素分泌细胞,但其分泌胰岛索的功能及对实验性糖尿病的治疗价值尚需要验证.目的:观察从大鼠的胰腺组织分离纯化干细胞在体外将诱导分化胰岛素分泌细胞后胰岛素mRNA的表达、分泌胰岛素的能力及对1型糖尿病模型鼠的治疗作用.设计:随机对照观察.单位:中日友好医院临床医学研究所.材料:实验于2004-08/2007-12在北京中日友好医院临床医学研究所病理生理研究室完成.选用8～10周龄雄性SD大鼠及雌性裸鼠各10只,均购自北京维通利华实验动物技术有限公司.实验过程中对动物的处置符合动物伦理学标准.尼克酰胺、链脲菌素均为Sigma公司产品,nestin抗体为BDBiosciences产品.大鼠胰岛素放射免疫检测试剂盒购自Linco research.胰岛索抗体及胰高糖素抗体为Santa Cruz产品.方法:应用nestin结合的免疫磁珠从SD大鼠胰腺导管细胞中分离和纯化干细胞,经体外扩增及诱导分化形成胰岛素分泌细胞.①采用RT-PCR法检测细胞分化过程中胰岛素mRNA的表达.②将干细胞经诱导分化形成的胰岛进行冰冻切片,采用免疫荧光染色观察胰岛素及胰商糖索阳性细胞表达.③对干细胞分化胰岛的胰岛素释放功能进行评价,放射免疫法测定上清中胰岛素检测干细胞分化形成的胰岛分泌胰岛素的能力.④鼠按220 mg/kg链脲菌素腹腔注射法制备为1型糖尿病模型,造模后随机摸球分为天然胰岛组及干细胞胰岛组,每组5只.将大鼠天然胰岛(SD大鼠分离)及干细胞分化形成的胰岛分别移植于糖尿病裸鼠左肾包膜下,观察移植后鼠尾静脉血糖变化.主要观察指标:①细胞分化过程胰岛索mRNA表达.②胰岛样结构中胰岛素及胰高糖素阳性细胞表达.③干细胞分化形成的胰岛分泌胰岛素情况.④移植后鼠尾静脉血糖变化.结果:①RT-PCR检测结果表明胰岛素mRNA的表达随诱导时间延长而明显升高.②免疫荧光染色结果显示胰岛结构的中间存在大量胰岛素阳性细胞,胰高糖素阳性细胞主要分布于胰岛样结构的周边.③干细胞分化的胰岛在高浓度葡萄糖刺激后明显缺乏快速相的胰岛索释放,而是表现细胞外上清中胰岛素浓度的缓慢升高.④天然胰岛组移植后3 d使血糖降低到10 mmol/L以下,至观察到60 d仍维持在正常水平:干细胞胰岛在移植后8 d使血糖降低至10 mmol/L以下,且在移植35 d后血糖逐渐升高并恢复到胰岛移植前的水平.结论:大鼠胰腺干细胞经体外扩增和诱导分化后可分化胰岛素分泌细胞,但对葡萄糖的刺激没有快速相的胰岛素分泌.将胰岛素分泌细胞移植到1型糖尿病模型裸鼠后可一定程度地改善糖代谢的紊乱.
展开▼
