胚胎干细胞R1诱导成胰岛素分泌细胞团的研究

目的 建立体外培养和扩增胚胎干细胞R1(ES-R1)的最佳条件;利用多种生长因子,优化培养条件,体外定向诱导ES-R1分化为胰岛素分泌细胞(IPCs).方法 以丝裂霉索-C处理的MEF为饲养层,培养液中加白血病抑制因子(LIF),ES-R1维持未分化状态并扩增,进行碱性磷酸酶染色.胚胎干细胞(ESCs)悬浮培养制成拟胚体(EBs),对培养至第4d的EBs开始定向诱导,依次加入无血清的ITS培养液,胰岛前体细胞增殖培养液和胰岛分化培养液,每种培养液内各培养6d,获得形态功能较成熟的IPCs.采用DTZ染色、免疫荧光染色、胰岛素刺激实验等方法对IPCs进行检测.结果 ESCs在饲养层细胞上呈克隆状生长,经碱性磷酸酶染色为阳性;EBs经3种诱导液诱导成三维立体的IPCs,IPCs被DTZ染成猩红色,胰岛素和胰高血糖素阳性表达,胰岛素刺激实验阳性.结论 ES-R1在体外培养时,用MEF做饲养层,培养液中添加一定浓度的LIF,可以最好地保持未分化状态并大量增殖.用分阶段添加不同生长因子的方法诱导ES-R1定向分化生成的IPCs在形态和功能上具有胰岛的特性.

DIFFERENTIATION OF EMBRYONIC STEM CELLS-R1 INTO INSULIN PRODUCING CELLS

Objective To define the best culture conditions of ES-R1 and explore the possibility and feasibility of differentiation of ES-R1 to IPCs in vitro with various of factors. Methods ESCs were cultured on the mitomycin C treated MEF feeder layer cells. The culture medium of ES-R1 was supplemented with 10μg/L LIF. ESCs were transferred into the ultra low adherent dishes in the absence of LIF and feeder layers for the formation of EBs. On day 4 of EBs formation, EBs were transferred to collagen-Coated dishes and serum free ITS A medium, pancreatic proliferation medium and pancreatic differentiation medium were added step by step, each for 6 days. During the differentiation from ESCs to IPCs, morphological methods, immunofluoresence staining, alkaline phosphatase staining, dithizong staining and insulin release test were used. Results Undifferentiated state was maintained by feeder cells and LIF. ESCs could form nested colonies with clear edge and smooth surface. Upon withdrawal of LIF and feeder layer, ESCs spontaneously formed into EBs in suspension. At the end of induction of differentiation, the three dimensional IPCs were formed. The induced IPCs were found to be stained crimson red by DTZ, insulin and glucagon immunohistochemistry staining positive, insulin release test positive.Conclusion Combination of LIF and MEF feeder layer culture may maintain the proliferation of ES cells without differentiation. Various factors used at different times, ES-R1 can differentiate into IPCs which possess ch