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目的检测Oct-4基因在BMSCs定向诱导分化中表观遗传学变化(即甲基化变化)。方法体外分离培养小鼠BMSCs,用成骨诱导剂(地塞米松,维生素C和β-甘油磷酸钠)诱导其向成骨细胞分化,通过间接免疫荧光染色和RT-PCR,检测OCT-4在小鼠BMSCs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BMSCs诱导分化前和分化后10 d的甲基化变化。结果 Oct-4在小鼠BM-SCs诱导前有表达,诱导10 d后表达下降;Oct-4在小鼠BMSCs诱导前未发生甲基化改变,诱导10 d后检测到其甲基化状态。结论 Oct-4在小鼠BMSCs诱导分化前后有甲基化状态的改变,且甲基化水平与表达呈负相关,提示Oct-4基因在小鼠BMSCs定向诱导分化中的表达下调可能是由于其启动子区高度甲基化的缘故。
Objective To detect the epigenetic changes (ie methylation changes) of Oct-4 gene in BMSCs induced differentiation. Methods BMSCs were isolated and cultured in vitro. Osteoblasts were induced by osteogenic agents (dexamethasone, vitamin C and β-glycerophosphate). Indirect immunofluorescence staining and RT-PCR were used to detect the expression of OCT-4 Mice BMSCs were induced to differentiate before and after differentiation. Methylation-specific PCR was used to detect the methylation of Oct-4 before and after 10-day differentiation of BMSCs. Results Oct-4 was expressed before the induction of BM-SCs in mice. The expression of Oct-4 was decreased 10 days after induction. Oct-4 was not methylated before the induction of BMSCs. The methylation status of Oct-4 was detected 10 days after induction. Conclusions The methylation status of Oct-4 before and after induced differentiation of BMSCs in mice has a negative correlation with the expression of Oct-4, which indicates that the down-regulation of Oct-4 gene in BMSCs induced differentiation may be due to Promoter hypermethylation of the reason.