Oct-4在骨髓基质细胞定向诱导分化中表观遗传学研究

METHYLATION OF OCT-4 GENE IN MOUSE BONE MESENCHYMAL STEM CELLS DURING DIFFERENTIATION IN VITRO

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 methylation level of Oct- 4 in bone mesenchymal stem cells during dif- ferentiation in vitro. Methods Mouse BMSCs were isolated and cultured in vitro, then induced them differ- entiate into osteoblasts by using osteogenic inducers such as dexamethasone, vitamin C and beta- glycero- phosphate. The level of OCT- 4 expression was detected by indirect immunofluorescence staining and RT - PCR before and after the induction of differentiation of mouse BMSCs; the methylation changes of Oct- 4 were also detected before and after the differentiation of mouse BMSCs by using of methylation- specific PCR. Results Indirect immunofluorescence staining and RT- PCR showed that expression of Oct- 4 BM- SCs was visible in mice before induction, whose expression level was decreased after induction of 10d. Methylation- specific PCR showed that methylation changes of Oct- 4 in mouse BMSCs did not occur be- fore induction, and methylation status were detected after induction of 10d. Conclusions Oct- 4 can induce differentiation in mouse BMSCs methylation status before and after the change, and the levels of methyla- tion and expression are negatively correlated. Downregulation of Oct- 4 gene in mouse BMSCs orientation induced differentiation may he due to hypermethylation of its promoter region,