摘要
目的探究启动子结合蛋白（C／EBPa）对于间充质干细胞（MSC）C3H10T1／2细胞成骨调节的分子机制。方法体外构建成骨分化模型，应用反向聚合酶链反应（BT-PCR）和免疫印迹检测C3H10T1／2细胞成骨分化时过氧化物酶体增殖物激活受体1（PPARγ）的mRNA和蛋白质的表达；通过构建干扰PPARlsiRNA的腺病毒载体及PPARγ拮抗剂G3335，观察PPARγ对于激活C／EBPa表达的作用。色质免疫沉淀分析（ChIP）检测PPARγ2位于C／EBPa启动子-1286bp／-1065bp区域内的结合位点并检测PPARγ与C／EBPa启动子-1286bp／-1065bp区段DNA甲基化和组蛋白高度去乙酰化的关系。结果C3H10T1／2细胞在成骨分化时，PPARγ的mRNA和蛋白质的表达呈先升高后下降的双时相反应。siRNA和G3335引起的PPARγ活性的下调抑制了C／EBPa在C3H10T1／2细胞成骨分化时的上调（对照组6．1mg／L，罗格列酮组13．0mg／L，罗格列酮＋G3335组11．0mg／L，P〈0．05）。DNA甲基化能减弱PPARγ2向-1286bp／-1065bp区段的结合能力。结论C3H10T1／2细胞在成骨分化时，PPARγ通过结合至C／EBPa启动子-1286bp／-1065bp区段来促进该基因的表达，阻抑了PPARγ与之结合，与PPARγ相关的蛋白去乙酰化酶抑制得以解除，从而下调了-1286bp／-1065bp区段的组蛋白乙酰化，C／EBPa启动子促进C3H10T1／2细胞成骨过程。
Objective To explore the molecular mechanisms of C/EBPa for regulating the osteogenesis of C3H10T1/2 cells. Methods We constructed an in vitro osteogenic differentiation model and investigated the mRNA and protein expression profile of PPARγ2 during C3H10T1/2 osteogenesis by reverse transcription-polymerase chain reaction （ RT-PCR ） and Western blot. Next we constructed an adenovirus vector loading siRNA against PPARγ2 and employed a specific antagonist of PPARγ, G3335, to examine the effect of PPARγ on activating the expression of C/EBPα. Then we performed ChIP to test PPARγ2 binding on - 1 286 bp/- 1 065 bp region of C/EBPα promoter and explore the role of PPARγ in the dependence of histone hypoacetylation on DNA methylation in - 1 286 bp/- 1 065 bp region of C/EBPα promoter. Results During the osteogenic differentiation, PPARγ mRNA and protein expression initially increased and then decreased in double-phase reactions, siRNA and G3335 induced down-regulation of PPARγ activity inhibited C/EBPα in C3H10T1/2 cells up-regulation of osteogenic differentiation. ChIP resuhs show that DNA methylation could weaken the binding section PPARγ2 to - 1 286 bp/- 1 065 bp capabilities. Conclusion This study provides a deeper insight into the molecular mechanisms underlying the osteogenesis of MSCs regulated by C/EBPα in synergy with PPARγ. PPARγ contributes to C/EBPα expression through binding on -1 286 bp/- 1 065 bp region of its promoter during the osteogenesis of C3H10T1/2 cells. And DNA hypermethylation in - 1 286 bp/- 1 065 bp region during the terminal stage of osteogenesis prevents PPARγ from binding on it so that PPARγ associated repression of HDAC1 is relieved to down-regulate the acetylationstatus of - 1 286 bp/- 1 065 bp region. Meanwhile DNA methylation and histone acetylation are linked by PPARγ for regulating the differentiation of MSCs.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2014年第3期227-231,共5页
National Medical Journal of China
