多种生长因子分阶段诱导小鼠胚胎干细胞分化为胰岛素分泌细胞

Differentiation of mouse embryonic stem cells into insulin-secreting cells induced by a 5-step model system

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 mouse embryonic stem （ES） cells to differentiate into insulin-secreting cells by means of a 5-step model system. Methods E14. 1 mouse ES cells were cultured in the presence of leukemia inhibitory factor （LIF） for 2 days （ step 1 ）, then the cells were cultured in hanging drops to form embryonic bodies （EBs） and the resulting EBs were cultured in suspension for 6 days in the presence of basic fibroblast growth factor bFGF （ step 2 ）. Subsequently the EBs were cultured in the medium containing glucagonlike peptide 1 （GLP-1）, hepatocyte growth factor （HGF）, nerve growth factor （NGF） and nicotinamide for 10 days （ step 3 ）. After that, the EBs were dissociated into single cells, and the cells were cultured in monolayer in the presence of GLP-1, betacellulin, activin A, bFGF and nicotinamide for 10 days （ step 4）. Finally, the cells were cultured in low-glucose medium containing nicotinamide for 4 days （ step 5 ）. Insulin and some other isletrelated genes expressions were investigated using RT-PCR and insulin expression was also investigated by DTZ- staining and immunohistochemistry. The percentage of insulin-secreting cells was evaluated by flowcytometry and insulin concentrations were measured by RIA. Results mRNA expression of insulin became visible at step 3 and more evident at step 5. Additionally, at step 5, mRNAs of glucagon, somatostatin, pancreatic polypeptide （ PP）, pancreatic duodenal homeobox 1 （ PDX-1 ） , beta-cell E box transactivator 2 （ Beta2 ） and neurogenin 3 （ Ngn3 ） were detected. DTZ-staining positive cells and insulin immunohistochemical staining positive cells were observed. The percentage of insulin-positive cells was （ 24.0 ± 2.5 ） % （ n = 6 ）. In the presence of 5.6 mmol/L and 25 mmol/L glucose, insulin concentrations were （0.05 ± 0.01 ） μg/L and （ 0. 13 ± 0. 02 ） μg/L respectively （ n = 6）. Conclusion E14.1 mouse ES cells can be induced to differentiate into insulin-secreting cells by the 5-step model system. Insulin-secreting cells can release insulin into culture medium when treated with glucose, and insulin concentrations increase with rising concentration of glucose.