Oct-4在精原干细胞定向诱导分化中甲基化的研究

Methylation of Oct-4 gene in the Spermatogonial Stem Cells of Mouse during Differentiation in Vitro

目的:检测Oct-4在小鼠精原干细胞(Spermatogonial Stem Cells,SSCs)诱导分化前后的表达及甲基化状态。方法:体外分离培养小鼠SSCs,用干细胞因子(stem cell factor,SCF)诱导其向精母细胞方向分化,通过间接免疫荧光染色和RT-PCR,检测Oct-4在小鼠SSCs诱导分化前后的表达;运用甲基化特异性PCR检测Oct-4在小鼠SSCs诱导分化前和分化后2d的甲基化状态。结果:间接免疫荧光染色和RT-PCR显示,Oct-4在小鼠SSCs诱导前有表达,诱导2d后表达下降;甲基化特异性PCR显示,Oct-4在小鼠SSCs诱导前未发生甲基化改变,诱导2d后检测到其甲基化状态。结论:Oct-4基因在小鼠SSCs定向诱导分化中的表达下调可能是其启动子区CpG岛甲基化的缘故。

Objective： Detect Oct-4 expression and the methylation leve in SSCs of mouse induced differentiation in vitro. Methods：The mouse spermatogunium stem cells （SSCs） were separated and purified, then induced with stem cell fac- tors （SCF） to differentiation in vitro. The immnofluorescence staining, RT-PCR and methylation-sensitive PCR techniques were uesd to detect Oct-4 expression and the methylation level in mouse SSCs induced. Results：The results showed that the Oct-4 expressed in primary cultured mouse SSCs, then decreased, and methylated after induction for 2 days. Conclusions： The methylation of CPG is lowed in Oct-4 gene promoter may contribute to the down-regulated expression of Oct-4 gene in mouse SSCs induced differentiation.