焦磷酸测序法检测OCT4启动子在大鼠骨髓源性肝干细胞分化中的甲基化变化  

Pyrosequencing Detection of OCT4 Promoter Methylation in Differentiation of Rat Bone Marrow-derived Liver Mesenchymal Stem Cells

作　　者：潘润华[1] 阿斯楞[1] 张茂[1] 利民[1] 张梓朗 廖彩仙[1] 

机构地区：[1]南方医科大学南方医院肝胆外科,中国广东广州510515

出　　处：《生命科学研究》2016年第2期113-118,共6页Life Science Research

基　　金：广东省科技计划项目(2010B080710169);国家卫计委重点临床专科资助项目(W0211WAI44)

摘　　要：骨髓间充质干细胞具有分化成为肝细胞的潜能已得到证实,但其分化的调控机理还不完全清楚。通过检测大鼠骨髓源性肝干细胞(rat bone marrow-derived liver stem cells,RBMLSCs)诱导分化为肝细胞过程中OCT4启动子甲基化的变化,来探讨启动子甲基化调控细胞分化的机制。RBMLSCs用25μg/L重组人肝细胞生长因子和0.1 nmol/L地塞米松进行诱导,在诱导之后不同时间点(0 d,7 d,14 d)提取细胞的DNA及RNA。用荧光定量PCR法检测白蛋白(albumin,Alb)和OCT4的m RNA表达水平,应用焦磷酸测序法定量检测OCT4基因的启动子中4个Cp G位点的甲基化变化。结果表明:在RBMLSCs分化过程中,Alb m RNA持续增高(P<0.05),但OCT4 m RNA表达在7 d及14 d明显减少(P<0.005);同时OCT4启动子上游的Cp G 1-2-3甲基化频率显著上升(P<0.001),但Cp G 4甲基化无明显变化。这些结果说明OCT4启动子高甲基化可能导致其m RNA表达减少,RBMLSCs多能性消失,但Cp G 4并不参与这一分化调控过程。It has been confirmed that bone marrow mesenchymal stem cells have the potential to differentiate into hepatocytes, however, the related regulation mechanism has not been elucidated. To explore the specific mechanism, OCT4 gene promoter methylation variation was detected in differentiation induction of rat bone marrow-derived liver stem cells （RBMLSCs） into hepatocytes in vitro. RBMLSCs were induced by 25 μg/L recombinant human hepatocyte growth factor and 0.1 nmol/L dexamethasone. DNA and RNA were extracted from cells at different time points （day 0, 7 and 14） after induction. Fluorescence quantitative PCR （FQ- PCR） was performed to detect OCT4 and albumin （Alb） mRNA expression. Pyrosequencing was performed to detect methylation variation of 4 CpG sites in OCT4 promoter. During cell differentiation, Alb mRNA ex- pression was increased significantly （P〈0.05）, but OCT4 mRNA was decreased significantly （P〈0.005） at day 7 and day 14. Methylation frequency of the first 3 CpG sites in OCT4 promoter upstream was increased significantly （P〈0.001）, but the 4th CpG site had no obvious methylation change. The results showed that the hypermethylation of OCT4 promoter may lead to the decline of its mRNA expression, disappearance of RBMLSCs pluripotency, but that the 4th CpG site might not be involved in the regulating process.

关 键 词：大鼠骨髓源性肝干细胞 OCT4 启动子甲基化 焦磷酸测序 

分 类 号：Q344[生物学—遗传学]