中文摘要：

【目的】利用C2C12成肌细胞探讨肌源性干细胞成脂过程与调控成脂和成肌分化的关键转录因子PPARγ（peroxisome proliferator-activated receptor gamma）、C/EBPα（CCAAT/enhancer binding protein alpha）和Myogenin启动子区甲基化的关系。【方法】分别用2%马血清和三联诱导剂诱导C2C12细胞成肌和成脂分化,在马血清促进的成肌分化第0、1、3和5天收集细胞进行姬姆萨染色观察肌管形成情况;在三联诱导的成脂分化第0、2、4、6和10天收集细胞进行油红O染色观察脂滴形成情况;提取成肌诱导第0、1、3、5天和成脂诱导第0、2、4、6天的RNA和DNA,分别采用qRT-PCR检测成肌和成脂分化相关基因的表达,采用重亚硫酸盐测序的方法检测PPARγ、C/EBPα和Myogenin启动子区甲基化的变化,并分析基因表达与甲基化状态的相关关系。【结果】①C2C12细胞经马血清诱导形成了多核肌管,表达成肌相关基因,但不表达脂肪特异性基因;三联诱导使C2C12细胞自主分化的肌管中沉积了脂滴,同时表达成脂和成肌相关基因;②重亚硫酸盐测序结果表明,在未分化的成肌细胞中,PPARγ基因启动子的甲基化水平是61%,在三联诱导第2、4和6天,其甲基化程度依次为49%、39%和42%,呈逐渐去甲基化趋势,与PPARγ基因转录负相关;在马血清诱导第3和5天,甲基化水平为56%和48%,与未分化的成肌细胞相比差异不显著,同时PPARγ基因的表达水平也没有显著变化;③Myogenin基因在马血清促进的成肌过程中甲基化水平不断降低（49%、42%、35%和34%）,转录水平急剧增加;在三联诱导过程中,Myogenin启动子的甲基化水平由0天的49%下降为第1天的37%、第3天的41%和第5天的38%,但下降幅度弱于马血清诱导的成肌过程,这一结果与降低的Myogenin转录上调相吻合;④C/EBPα基因在未分化的C2C12细胞中呈低甲基化状态,甲基化程度仅为1.6%,在成肌分化和脂肪沉积过程中均未发生?

英文摘要：

[Objective] This experiment was designed to investigate whether DNA methylation of adipogenic promoters （peroxisome proliferator-activated receptor gamma （PPART） and CCAAT/enhancer binding protein alpha （C/EBPa）） and myogenic promoter （myogenin） was involved in C2C12 myoblasts intracellular lipid accumulation. [Method] C2C12 cells were treated, respectively, by 2% horse serum and an adipogenesis cocktail which typically contains 3-isobutyl-methylxanthine, dexamethasone and insulin （MDI）. In order to observe the formation ofmyotubes, cells were incubated for 0 d, 1 d, 3 d, and 5 d by 2% horse serumand then were collected to perform Giemsa staining. To confirm the lipid accumulation, cells treated by adipogenesis program for 0 d, 2 d, 4 d, 6 d and 10 d were fixed to conduct Oil Red O staining. Genome DNA and total RNA were extracted from C2C12 cells during the myogenesis differentiation and intracellular lipid accumulation. Real-time quantitative reverse transcription PCR was used to detect the mRNA levels of myogenic and adipogenic related genes. The bisulfite sequencing was adopted to explore the sequential DNA methylation changes in PPART, C/EBPct and myogenin promoter regions, and the relationship between DNA methylation and gene expression was analyzed. [Result] After horse serum treatment, the morphology of multinucleated myotubes and up-regulation of myogenic genes were displayed in C2C12 cells, but the adipocyte-specific genes showed no significant changes. The adipogenesis cocktail induced the intracellular lipid accumulation during automatical differentiation into myotubes of C2C12 cells, and both the myogenic and adipigenic genes were up-regulated. The bisulfite sequencing showed that the methylation level of PPARy gene in undifferentiated myoblasts was 61%, and progressively demethylated upon adipogenic differentiation with methylation levels of 49%, 39% and 42% on d 2, 4 and 6, which was accompanied by an increase of mRNA level of PPART. But during the horse serum treatment, there w