小鼠胚胎干细胞定向诱导为胰岛样细胞过程中印记基因变化

Expression of imprinted genes during the course of differentiation from mouse embryonic stem cells to islet-like cells in vitro

目的探讨体外诱导对小鼠胚胎干细胞株SF1-G印记基因Kcnq1、Cdkn1c的影响。方法体外诱导小鼠胚胎干细胞SF1-G向胰岛样细胞分化,RT-PCR和细胞免疫荧光检测胰岛细胞标志基因表达;聚合酶链式反应-限制性内切酶片段长度多态性(PCR-RFLP)检测印记基因Kcnq1、Cdkn1c在分化各阶段细胞中的亲本表达情况。结果胰岛细胞标志性基因Insulin、Glucagon、Somatostatin、IAPP、Glut2在诱导分化后的细胞都有表达;分化终末细胞能表达胰岛细胞特异性蛋白胰岛素、C肽及Somastatin;PCR-RFLP检测分析显示,体外诱导分化后细胞的印记基因Kcnq1、Cdkn1c呈双等位基因表达。结论胚胎干细胞经体外诱导培养可定向分化为胰岛样细胞;用此种分化方法可导致胚胎干细胞印记基因表达的改变,出现印记丢失。

Objective To study the effects of in vitro inducement on the expression of SF1-G imprinted genes,Kcnq1 and Cdkn1c during the course of differentiation from mouse embryonic stem（ES） cells to islet-like cells.Methods Mouse ES cells were induced to differentiate into islet-like cells in vitro.The expression of islet specific markers was tested by RT-PCR or immunofluorescence.RT-PCR/RFLP was used to test the imprinted genes parental expression in cells at different stages.Results Islet specific genes,such as Insulin,Glucagon,Somatostatin,IAPP and Glut2,were expressed in differentiated cells.The proteins of insulin,C-peptide and Somastatin were expressed in the final stage cells.Imprinted gene Kcnq1 and Cdkn1c were biallelicly expressed in islet-like cells.Conclusions Mouse ES cells can be successfully induced into islet-like cells in vitro.Gene imprinting status of Kcnq1 and Cdkn1c may be changed in differentiated cells（causing loss of imprinting） during the in vitro inducement.