体外胰岛细胞共培养及化学因子联合诱导NOD鼠源性诱导性多能干细胞分化为胰岛素分泌细胞的研究

Experimental study on combination of islet cells and chemical factors to induce pluripotent stem cells derived from NOD mouse model who can differentiate to insulin-producing cells in vitro

目的建立更为高效的NOD鼠体外胰岛素分泌细胞诱导体系。方法化学因子分步加入培养基,将大鼠胰岛细胞以半透膜相隔与NOD鼠源性诱导性多能干细胞(NOD-iPSCs)共培养约20d,检测胰岛素分泌细胞的相关功能,并与纯化学因子诱导方法进行对比。结果诱导后成团生长,双硫腙染色呈棕红色,RT-PCR显示胰十二脂肠同源异型盒基因1(PDX-1)从第8天起开始表达,并逐渐增强。免疫荧光染色显示细胞表达胰岛素及C-P,联合组及纯化学因子组诱导培养每隔4d检测至20d,高糖刺激后两组胰岛素分泌均呈逐渐升高趋势。结论 NOD-iPSCs能够在体外诱导生成胰岛素分泌细胞。联合培养体系提供大鼠胰岛细胞功能因子可加速分化,提高效率,可能为一种更高效的诱导方法。

Objective To build a highly efficient approach to induce iPSCs derived from NOD mouse model to differentiate into insulin producing cells （IPCs） in vitro. Methods Chemical factors were steped to join in culture, then rat islet cells were separated by transwell insert, and co-cultured with NOD mouse-derived iPSCs （NOD-iPSCs） for 20 days . Glucose-simulated insulin secretion was tested and the results also were compared with the group of only using chemical factors. Results At about 14 days after beginning differentiation, morphological changes of forming clusters gradually were observed following induction under an inverted microscope. And DTZ-stained cell clusters formed. The mRNA（PDX- 1） associating with pancreatic progenitor cells was expressed by RT-PCR technology from day 8 to day 20. Insulin and C-peptide in cells was examined by immunocytochemistry. After beginning differentiation, we tested the produced insulin by high glucose stimulating every 4 days. The results of co-culture-chemical factors group and chemical factors group were 0 ng/ml and 0 ng/ml, （1.67＋0. 19） ng/ml and （0.45＋ 0. 23） ng/ml, （2.97＋0.26） ng/ml and （2.34＋0. 11） ng/ml, （3.07＋0.17） ng/ml and （2.59＋0.24） ng/ml, （3.16＋0.27） ng/ml and （2.67＋0.19） ng/ml（P〈0.05）. Conclusion We developed a method for stepwise differentiation of NOD-iPSCs into IPCs combining with islet cells in co-culture and chemical factors in vitro. These IPCs can store and release insulin in response to glucose. This combination method seems to be a more effective protocol.