脂质代谢是人体生命活动的基本问题之一，脂肪细胞的分化机制及脂质合成过程中的DNA甲基化调控研究是细胞生理学探索的重要领域。目前已经证明脂肪细胞的分化与糖、脂代谢与机体能量平衡关系密切，其调控失常与人类多种疾病如肥胖症、糖尿病、脂肪肝、高脂血症及乳腺癌等密切相关。因此，对脂肪细胞分化机制及其脂质合成的关键靶基因的DNA甲基化调控机制的研究，对于探讨肥胖等相关疾病的过程具有重要的理论意义。本研究是在前期研究的基础上，利用3T3-L1前脂肪细胞和小鼠白色脂肪组织，对前脂肪细胞分化的DNA甲基化调控机制进行了初步探索。利用生物信息学方法证明调控脂肪分化和脂质合成相关基因启动子区域大都含有CpG岛。甲基化敏感性酶切实验证明在脂肪细胞分化的过程中，随着脂肪细胞的分化成熟其基因组DNA甲基化水平提高。RT-PCR检测发现甲基转移酶在前脂肪细胞的分化过程中均呈现出分化前期表达较高，而随着脂肪细胞的分化成熟其基因表达水平逐渐下降的趋势。通过BSP法对脂肪分化关键基因C/EBPα启动子区DNA甲基化水平进行检测，发现脂肪细胞成熟后C/EBPα启动子区DNA甲基化程度高于未分化时的状态。荧光素酶报告实验证明甲基化后C/EBPα基因启动子转录活性高于非甲基化状态。同时，油红染色实验证明DNA甲基转移酶抑制剂AzaD对脂肪细胞分化和脂肪合成具有一定的抑制作用。AzaD抑制脂肪细胞分化关键基因C/EBPα和脂肪合成基因FAS，LPL的转录表达，并呈现出浓度依赖效应。此外，甲基化敏感的限制性指纹谱技术筛选出脂肪细胞分化前后两个甲基化敏感基因CMTM2b和SRRM4，该基因在前脂肪细胞的分化过程中均呈现出分化前期表达较高，而随着脂肪细胞的分化成熟其基因表达水平逐渐下降的趋势。总之，本研究揭示了前脂肪细胞分化的DNA甲基化调控的可能机制，从而为阐明肥胖等相关疾病的发生过程提供线索并且为药物治疗肥胖提供新的靶点。

Lipid metabolism plays a vital role in life activities. Mechanisms of preadipocyte differentiation and lipid synthesis regulated by DNA methylation are an important field of cell physiology. Now it is proved that preadipocyte differentiation is inseparable related with glucose and lipid metabolism as well as energy balance. The disorder of preadipocyte differentiation is closely related with many diseases, such as obesity, diabetes, fatty liver, hyperlipemia and breast cancer. Therefore, the investigation of the mechanism underlying DNA methylation regulation of preadipocyte differentiation is crucial for the understanding of obesity and related diseases pathology. On the basis of the previous studies, we evaluated the mechanism of DNA methylation regulation of preadipocyte differentiation using 3T3-L1 preadipocytes and mouse white adipose tissue. First, we found many preadipocyte differentiation and adipogenesis lipogenic gene promoter region containing CpG islands through bioinformatics methods. It is showed that with the differentiation of preadipocyte, the genomic DNA methylation level increased significantly by methylation-sensitive restriction enzyme digestion assay. Consistent with this result, the transcription expression level of DNA methyltransferases gradually decreased during the differentiation of preadipocyte. Furthermore, the DNA methylation levels of differentiation key gene C/EBPα promoter of adipocyte are higher than the state of preadipocyte detected by BSP method. The results of luciferase assay suggested that the transcription activity of methylated C/EBPα gene promoter is higher than non-methylated plasmid. In addition, DNA methyltransferase inhibitor AzaD has a potent anti-adipogenic effect in 3T3-L1 cells due to the inhibition of adipocyte differentiation and adipogenesis. At the molecular level, AzaD inhibited the expression of C/EBPα, as well as FAS and aP2 in dose-dependent manner. Furthermore, we identified 2 new methylation-sensitive genes named CMTM2b and SRRM4 using MSRF screening method that exhibited possible changes toward hypermethylation in adipocyte compared with their status in preadipocyte. Taken together, our results show the possible mechanism underlying DNA methylation regulation of preadipocyte differentiation, and those findings provide a novel clue for the understanding of obesity pathology and new target for obesity therapy.