摘要
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： The deoxycytidine analog 5-Aza-2′-deoxycytidine（5-Aza-dC） has been widely used as a DNA methylation inhibitor to experimentally induce gene expression and cellular differentiation.The aim was to investigate role of 5-Aza-dC-induced DNA methylation involved in differentiation mouse ES cells into germ cells.Methods： To measure the sensitivity to the toxic effect of the drug,mouse ES cells-derived embryoid bodies（EBs） were exposed to various concentrations of 5-Aza-dC at 0.05μmol/L^1μmol/L drug concentration.The expression of Dnmt3a and Dnmt1 were detected by RT-PCR,real-time PCR and Western blot,CpG island methylation status of germ cell-specific gene Mvh was evaluated by methylated specific PCR（MSP） combined with sequence analyse.Results： When exposed to 5-Aza-dC,the morphology and phenotype of EBs were changed,with increasing expression of Dnmt3a and Dnmt1 in mRNA and protein levels.While the germ cell-specific gene Mvh were demethylated in EB cells,but not in mES cells after 5-Aza-dC treatment.Conclusions： The effect of 5-Aza-dC may be directly affecting mouse embryonic stem cells-derived EBs through the covalent binding of DNA methyltransferase,the expression of Mvh gene is under the control of an epigenetic mechanism mediated by DNA methylation by trapping of Dnmt1 and Dnmt3a.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2011年第11期44-50,共7页
China Biotechnology
基金
国家自然科学基金(81100471)
高等学校博士学科点专项科研基金(20090001120130)
深圳市科技计划项目(20110421005)
深圳市科技计划重点项目(201001014)资助项目
深圳市科技研发资金基础研究计划(JC201005260214A)
