三种不同细胞培养方式对体外小鼠胚胎干细胞分化为胰岛素分泌细胞诱导效果的比较

目的：比较胚胎体、胚胎体-细胞单层和细胞单层3种细胞培养方式对小鼠胚胎干细胞（ES细胞）分化为胰岛素分泌细胞的诱导效果方法：应用胰高血糖素样肽-1（GLP-1）、β细胞素(betacellulin)、激活素A（activin A）、碱性成纤维细胞生长因子（bFGF）和尼克酰胺（nicotinamide），分别以胚胎体、胚胎体-细胞单层和细胞单层3种细胞培养方式诱导小鼠ES细胞30 d后，应用RT-PCR、双硫腙（DTZ）染色和免疫组化检测分化细胞胰岛素表达，以流式细胞仪检测胰岛素阳性细胞百分比结果：3种细胞培养方式诱导的分化细胞均可见DTZ染色和胰岛素免疫组化染色阳性细胞，RT-PCR检测到胰岛素和其它一些胰岛相关基因mRNA表达，胚胎体-细胞单层方式胰岛素mRNA表达最强，胚胎体方式次之，细胞单层方式最弱。胚胎体-细胞单层方式的胰岛素阳性细胞百分比高于胚胎体方式（P＜0.01），后者又高于细胞单层方式(P＜0.01）结论：采用胚胎体-细胞单层方式诱导可更好促进小鼠ES细胞分化为胰岛素分泌细胞。

Comparison of the effects on differentiation of mouse embryonic stem cells into insulin-secreting cells among three cell culture protocols

AIM: To compare the effects of three different cell culture protocols: embryonic body(EB) formation,EB formation-monolayer and monolayer on differentiation of mouse embryonic stem(ES) cells into insulin-secreting cells.METHODS: E14.1 mouse ES cells were treated with GLP-1,betacellulin,activin A,bFGF and nicotinamide by using EB formation,EB formation-monolayer and monolayer culture protocol respectively for 30 days,then insulin expression was examined by RT-PCR,DTZ-staining and immunohistochemistry.The percentage of insulin-secreting cells was evaluated by flow cytometry.RESULTS: DTZ-staining positive cells and insulin immunohistochemical staining positive cells were observed in the differentiated cells for all the three groups.mRNAs of insulin and some other islet-related genes were detected,insulin expression was the strongest in EB formation-monolayer,and the weakest was in monolayer.The percentage of insulin-positive cells of the differentiated cells in the EB formation-monolayer group was higher than that in the EB formation group(P<0.01),the latter was higher than that in the monolayer group(P<0.01).CONCLUSION: Among the three cell culture protocols,EB formation-monolayer is the most effective approach in the induction of mouse ES cells to differentiate into insulin-secreting cells.