论文部分内容阅读
目的以一种5个阶段的诱导方法诱导小鼠胚胎干(ES)细胞分化为胰岛素分泌细胞。方法以一种5个阶段的、包含胰高血糖素样肽1(GLP-1)、肝细胞生长因子(HGF)、神经生长因子(NGF)、β细胞素(betaceuulin)、激活素A(activin A)、碱性成纤维细胞生长因子(bFGF)和尼克酰胺等7种生长因子的诱导方法诱导ES细胞30d,应用RT-PCR、双硫腙染色和免疫组化检测胰岛素表达,以流式细胞仪检测胰岛素阳性细胞百分比,用RIA法测定培养液胰岛素水平。结果分化细胞中可检测到胰岛素和一些其他胰岛相关基因mRNA表达。双硫腙染色和胰岛素免疫组化染色阳性。分化细胞胰岛素阳性细胞百分比为(24.0±2.5)%(n=6)。在5.6mmol/L和25mmol/L葡萄糖浓度作用下,培养液胰岛素水平分别为(0.05±0.01)μg/L和(0.13±0.02)μg/L(n=6)。结论应用上述分阶段诱导方法可将小鼠ES细胞诱导分化为胰岛素分泌细胞,在葡萄糖作用下该细胞能释放胰岛素到培养液中,胰岛素分泌水平随着葡萄糖浓度的增高而增高。
Objective To induce the differentiation of mouse embryonic stem (ES) cells into insulin-secreting cells by a five-stage induction method. METHODS: A five-stage model of glomerulonephritis with GLP-1, hepatocyte growth factor (HGF), nerve growth factor (NGF), betaceuulin, activin ES cells were induced by 7 kinds of growth factors, including basic fibroblast growth factor (bFGF) and nicotinamide for 30 days. The expression of insulin was detected by RT-PCR, dithizone staining and immunohistochemistry. Flow cytometry Instrument detection of insulin-positive cell percentage, RIA determination of insulin solution level. As a result, mRNA expression of insulin and some other pancreatic islet-related genes was detected in differentiated cells. Dithizone staining and insulin immunohistochemical staining were positive. The percentage of differentiated cells in insulin-positive cells was (24.0 ± 2.5)% (n = 6). Insulin concentrations in culture medium were (0.05 ± 0.01) μg / L and (0.13 ± 0.02) μg / L, respectively (n = 6) under the effects of 5.6 mmol / L and 25 mmol / L glucose. Conclusion The above-mentioned staged induction method can induce mouse ES cells to differentiate into insulin-secreting cells. Under the action of glucose, the cells can release insulin into the culture medium, and the insulin secretion level increases with the increase of glucose concentration.