PcG蛋白EZH2在全反式维甲酸诱导的小鼠iPS细胞系分化过程中的作用

目的探讨组蛋白甲基转移酶EZH2在小鼠iPS细胞系IP14D-1向生殖细胞分化过程中的作用。方法将小鼠iPS细胞系IP14D-1通过悬浮培养分化形成拟胚体(iEBs)作为向生殖细胞分化的启动步骤,1μmol/L全反式维甲酸(atRA)持续诱导iEBs 2、4和7 d,采用实时RT-PCR、荧光定量RT-PCR、免疫荧光检测atRA处理前后EZH2和生殖细胞分化标志性基因Stra8在iEBs中的表达。结果在atRA诱导IP14D-1来源的iEBs中,EZH2表达在诱导后2 d达到高峰,伴随atRA诱导,EZH2表达减弱;而生殖细胞分化标志性基因Stra8其变化特点与EZH2相反,无atRA诱导时EBs表达Stra8较弱,伴随atRA诱导,Stra8表达增强。EZH2和Stra8阳性信号分别定位于胞膜和胞质,其变化特点与PT-PCP结果相一致。结论 atRA诱导使EZH2低水平表达时期提前出现,伴随Stra8增强表达,启动了小鼠iPS细胞向生殖细胞的分化进程。

The role of PcG protein Ezh2 in the process of differentiation of mouse induced pluripotent stem cells introduced by atRA

Objective To investigate the role of Histone Methyltransferase EZH2 when mouse induced pluripotent stem (iPS) cell line IP14D-1 differentiates into primordial germ cells(PGCs) in vitro. Methods IP14D-1-derived EBS were maintained in culture medium containing 1μM atRA for 2d, 4d and 7d, respectively. RT-PCR、Western blot and immunofluorescence were performed to detect the expression changes of Ezh2 and Stra8 in the atRA processing IP14D-1-EBs around. Results The expression features of genes showed that Ezh2 mRNA rapidly reached peak value in 2d, and with the induction of prolonged expression decreased; In contrast, Stra8 expression increased when the EBs induced with atRA to extend. Immunofluorescence showed positive signals of Ezh2 and Stra8 were located on the membrane and in the cytoplasm, this result was consistent with the RT-PCR. Conclusions The EZH2 appeares earlier in IP14D-1-derived EBS and is due to the induction of the atRA, and the expression of Ezh2 and Stra8 is not coordinated. Low expression of EZH2 may occur earlier in the differentiation of PGCs with atRA introduction, the enhanced expression of Stra8 may start the differentiation of IP14D-1 to the germ cell.