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目的:探讨小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向生殖细胞(Embryonic germcells,EG)分化过程中5-杂氮-2’-脱氧胞苷(5-Aza-2’-deoxycytidine,5-Aza-dC)对DNA甲基化转移酶Dnmt1和Dnmt3a及生殖细胞特征基因Mvh表达变化的DNA甲基化调控机制。方法:将mES细胞分化形成拟胚体(embryoid bodies,EBs)作为向生殖细胞分化的启动步骤,采用不同浓度(0.05μmol/L,0.1μmol/L,0.5μmol/L,1μmol/L,3μmol/L)处理EBs,RT-PCR实时荧光定量RT-PCR和Western blot分别检测检测在5-Aza-dC处理前后Dnmt1和Dnmt3a在ES细胞和EBs中的表达,甲基化特异性PCR(MSP)检测原始生殖细胞分化特征基因Mvh启动子甲基化状态。结果:5-Aza-dC的浓度在0.05μmol/L～1μmol/L之间时,EBs保持较高的存活率而EBs的形态明显发生了变化;5-Aza-dC处理后,Dnmt1和Dnmt3a在EBs中mRNA表达量明显降低,其变化特点与WB结果相一致。MSP和测序结果显示,Mvh启动子区表现为部分甲基化,5-Aza-dC处理后的4d EBs中Mvh CpG岛有4个CG位点发生突变,而mES细胞中未见突变。结论:EBs经5-Aza-dC处理后,Dnmt1和Dnmt3a的表达明显下调;同时,Mvh启动子发生部分甲基化,有可能启动了向生殖细胞的分化进程。
Objective: To investigate the role of 5-Aza-2’-deoxycytidine (5-Aza-2’-deoxycytidine) in the process of mouse embryonic stem cells (mESCs) differentiating into embryonic germ cells Aza-dC) on DNA methyltransferase Dnmt1 and Dnmt3a and germ cell gene Mvh expression changes in DNA methylation regulation. METHODS: The mES cells were differentiated to form embryoid bodies (EBs) as initiating steps to differentiate into germ cells. The cells were treated with different concentrations (0.05μmol / L, 0.1μmol / L, 0.5μmol / L, 1μmol / L, 3μmol / L), the expression of Dnmt1 and Dnmt3a in ES cells and EBs before and after treatment with 5-Aza-dC were detected by RT-PCR and Western blot respectively. Methylation-specific PCR (MSP) Primitive germ cell differentiation characteristic gene Mvh promoter methylation status Results: The EBs maintained a high survival rate and the morphology of EBs changed obviously when the concentration of 5-Aza-dC was between 0.05μmol / L and 1μmol / L. After treatment with 5-Aza-dC, Dnmt1 and Dnmt3a EBs mRNA expression was significantly reduced, the characteristics of its change and the results of WB consistent. The results of MSP and sequencing showed that the Mvh promoter region was partially methylated. In the 4d EBs treated with 5-Aza-dC, 4 CG sites of Mvh CpG island were mutated and no mutation was found in mES cells. CONCLUSION: The expression of Dnmt1 and Dnmt3a in EBs treated with 5-Aza-dC is significantly down-regulated. At the same time, partial methylation of Mvh promoter may initiate the process of germ cell differentiation.