人脂肪基质细胞向胰岛素分泌细胞的分化诱导

目的:体外分离培养人脂肪来源基质细胞（hADSCs），并定向诱导hADSCs向胰岛样细胞分化，探讨hADSCs分化为胰岛素分泌细胞的潜能。方法:从脂肪抽吸物中分离hADSCs，利用碱性成纤维细胞生长因子(bFGF)、上皮细胞生长因子(EGF)、尼克酰胺和exendin-4等因子诱导hADSCs向胰岛样细胞分化。采用RT-PCR检测诱导前后胰岛素、胰十二指肠同源性盒因子(Pdx-1)、葡萄糖转运因子2(Glut-2)和胰高血糖素(glucagon)基因的表达；双硫腙染色鉴定胰岛样细胞团；免疫荧光染色检测诱导后细胞胰岛素和Pdx-1的表达；ELISA检测诱导后细胞在低糖和高糖环境中胰岛素的分泌量。结果:诱导7 d左右细胞由长梭形逐渐变成多边形，并且细胞开始呈岛状聚集，21 d左右形成胰岛样细胞团。双硫腙染色阳性；RT-PCR显示，诱导后细胞表达胰岛素、Pdx-1、Glut-2和glucagon基因；免疫细胞化学显示，诱导后细胞表达胰岛素和Pdx-1；葡萄糖刺激实验显示胰岛素的释放，并且随着葡萄糖浓度增加胰岛素的释放量也增加。结论:体外hADSCs具有分化为胰岛素分泌细胞的潜能，为将来应用于临床移植治疗糖尿病奠定了基础。

In vitro differentiation of human adipose tissue-derived stromal cells into insulin-producing cells

Objective To isolate human adipose tissue-derived stromal cells(hADSCs) from the adipose tissue in vitro,and to induce the differentiation of hADSCs into insulin-producing cells,and to study the potential of hADSCs to differentiate into insulin-producing cells.Methods hADSCs were isolated from the adipose tissue.The hADSCs were differentiated to islet-like cells using basic fibroblast growth factor(bFGF),epidermal growth factor (EGF),nicotinamide and exendin-4.RT-PCR analysis was used to detect the expressions of genes in differentiation,such as pancreatic and duodenal homeobox 1(Pdx-1),insulin and glucose transporter 2(Glut-2);the differentiated cells were tested by Dithizone staining;Immunocytochemical analysis was performed to determine the insulin and Pdx-1;ELISA was done to assay the insulin secretion in response to glucose stimulation.Results hADSCs became polygon from fibroblast-like about 7 d after treated with the induced medium,and began to gather and started to form cell aggregates,the islet-like cell clusters were formed at 21 d.Dithizone staining was positive;RT-PCR analysis showed the expressions of the insulin,Pdx-1,Glut-2 and glucagon in the differentiated cells.Immunocytochemical analysis showed that the induced cells expressed insulin and Pdx-1.These cells secreted insulin in response to glucose stimulation,and the levels of insulin secretion were increased with the increasing of glucose concentrations.Conclusion hADSCs isolated from adipose tissues can be induced to differentiate into insulin-producing cells,and they can be used as a potential source for transplantation in the patients with diabetes mellitus.