启动子区DNA甲基化在肥胖瘦素基因表达调控中的作用研究

The mechanism of highly expressed leptin and leptin resistance in obesity is far from being fully understood. Recent reports demonstrate that the increased leptin expression in obesity may be associated with the CpG island methylation of its promoter, however their causual relationship and specific regulation mechanism need to be clarified. In this project, we plan to investigate the changes in methylation of the leptin promoter and associated methylated CpG binding protein 2 (MecP2), enzymes of epigenetic modification and histone modification (methylation and acetylation) in diet induced obese (DIO) mice, by using chromatin immunoprecipitation (ChIP) and PCR methods. Furthermore, DNA methyltransferase (DNMT) inhibitor and methy-donor (vitamin B12 and folic acid) will be used to reduce and promote DNA methylation respectively in DIO mice, so as to clarify the effects of the leptin promoter methylation on its expression and obesity pathogenesis. As well, DNMT gene knockout mice are to be included for study. In addition, the effects of obesity in pregnancy on the changes in the leptin promoter methylation and obesity pathogenesis in offspring are also to be studied by using DIO mice. This project will be helpful and meaningful for understanding the epigenetics of the leptin expression and resistance and exploring the new preventive and theraputic methods in obesity.

肥胖时瘦素表达增加与抵抗的发生机制尚不清楚。近来的研究显示，肥胖瘦素表达异常与基因启动子DNA甲基化可能相关，但是具体的因果关系和调控机制尚不清楚。本项目拟以小鼠为对象，采用染色质免疫沉淀（ChIP）-PCR等技术，观察肥胖状态下瘦素基因启动子区DNA甲基化及关联的甲基化CpG结合蛋白2（MeCP-2）、DNA甲基转移酶（DNMTs）酶的变化，进而采用DNMTs抑制剂和甲基供体干预小鼠；探讨启动子区DNA甲基化改变及关联的MeCP-2、表观修饰酶对瘦素表达调控和肥胖发生的影响。项目对于揭示肥胖瘦素表达的表观遗传调控机制，对于探索新的肥胖干预治疗措施，具有重要意义。

关于肥胖状态下瘦素表达异常以及瘦素抵抗发生的机制，仍不十分清楚。近年来的研究显示，表观遗传修饰与基因表达有关；肥胖时瘦素基因启动子区CpG位点发生了甲基化改变。本研究在前期工作基础上，以高脂饲料诱导肥胖小鼠为对象，采用亚硫酸盐修饰直接测序（BSP）法和染色质免疫沉淀（ChIP）-PCR等技术，观察肥胖发生过程中瘦素基因启动子区CpG岛甲基化改变以及其相关联的甲基化结合蛋白2（MBD2）、DNA甲基转移酶（DNMTs）的变化及其相互作用；进而采用DNMT酶抑制剂5-氮-2脱氧胞苷（5-Aza）、甲基供体维生素B12和叶酸进行干预，观察CpG岛DNA甲基化改变以及相关联的MBD2的变化，以期阐明肥胖时瘦素基因启动子区DNA甲基化修饰在基因表达和肥胖发生发展过程中的作用。. 肥胖小鼠发生过程中瘦素基因表观修饰研究显示：与正常脂饲料喂养小鼠相比，高脂饲料喂养小鼠脂肪组织瘦素mRNA表达在开始喂养后的第4、8、12和18周均显著高于对照组；然而瘦素基因启动子区CpG甲基化程度在第8周时降低并伴随MBD2和DNMT1结合的减少，第12和18周时启动子区CpG甲基化程度升高并伴随MBD2和DNMT3A和DNMT3B结合增多；另外启动子区结合的RNA聚合酶II在8周时增加而18周时减少。. 甲基供体和DNMT抑制剂干预研究显示，高脂饲料中补充维生素B12和叶酸干预后，自第12周开始体重明显低于正常饲料组；5-Aza注射小鼠体重显著低于高脂饲料生理盐水注射组；肥胖发生后，注射5-Aza 1个月后小鼠体重未见明显变化。脂肪组织瘦素基因mRNA的表达，与正常脂饲料喂养组相比，除了在高脂饲料诱导肥胖组显著增加以外，其他各组均无明显变化。瘦素基因启动子区CpG位点总甲基化程度在B12和叶酸以及5-Aza注射组均较正常脂饲料组显著升高；肥胖发生后，5-Aza注射降低了基因启动子区CpG位点的甲基化程度。瘦素基因启动子区HDAC1的结合研究发现，高脂饲料中B12和叶酸的补充降低了其结合量。. 结论：在肥胖发生过程中瘦素基因启动子区DNMTs结合随时间而变化；DNMTs活性在肥胖瘦素基因表达调控中可能起着重要作用。