细胞周期调控相关基因DNA甲基化及组蛋白乙酰化与氟中毒关系研究

The endemic fluorosis, which is widely prevalent in our province, has been consecutively listed in our country's "Twelfth Five-year Plan" and "Healthy China 2020" Strategic Plan as an important prevented disease. The pathogeny of endemic has been found out, however, the pathogenesis is not clear, which threatens people's health and hinders the development of economy and society. The endemic fluorosis has become a public concern which needs to be solved promptly. This research is based on epigenetics theory and the latest technology, which is combined with the locale of the endemic fluorosis caused by coal in our province and in vitro test. The research focuses on the characteristics of osteoblast being activated and starts from cell cycle regulation related gene（cyclinD1、CDK4、p16、p21）.This research makes full use of advanced bisulfate-sequencing PCR,methylation sensitivity-high resolution melting curve analyse and chromatin immuno-precipitation . The research tries to discuss fluorosis pathogenesis from the overall (human) and in vitro (cell) two experimental system, and the cell cycle regulation related gene epigenetics regulation , mRNA transcription and protein expression in three level, and uses inhibitor vitro intervention. Its purpose is to explore fluorosis mechanism with a new perspective in epigenetics and to provide creative ideas and directions for the prevention and therapy of fluorosis.

广泛流行在我省的地方性氟中毒（简称地氟病）是持续列入我国"十二五"和"健康中国2020行动计划"的重点防治疾病，该病虽然病因清楚，但致病机制未明，严重危害居民健康，影响经济社会发展，是我省亟待解决的重大公共卫生问题。本项目利用当前国际国内研究热点-表观遗传学的理论及最新技术，结合我省燃煤污染型氟中毒病区现场和体外试验，围绕成骨细胞被激活的特点，以细胞周期调控相关基因（cyclinD1、CDK4、p16、p21）为切入点展开研究。采用先进敏感的硫化测序PCR、甲基化敏感性高分辨率熔解曲线分析、染色质免疫共沉淀等方法，从整体（人体）和体外（细胞）两个实验体系，及细胞周期调控相关基因表观遗传学调控、mRNA转录及蛋白表达三个水平来探讨氟中毒致病机制，并利用抑制剂进行体外实验干预。希望能从全新视角-表观遗传学角度探索氟中毒作用机制，并为寻找氟中毒治疗手段提供新的思路和方向。

氟中毒病因清楚，但其病变复杂多样，致病机制困扰医学界多年。成骨细胞增殖活跃和骨转换加速在氟骨症病变中是一个发生较早、并起主导作用的环节。本项目利用贵州省燃煤污染型氟暴露病区现场和人群资源，结合体外实验，围绕氟骨症成骨细胞被激活的特点，以细胞周期调控相关基因为切入点，有针对性地选择细胞周期G1期重要的正负调控因子（cyclinD1、CDK4、p16、p21）展开氟表观遗传学研究。研究发现：① 氟可致细胞周期正性调控因子cyclinD1、CDK4 mRNA转录和蛋白表达增强，负性调控因子p16、p21 mRNA转录和蛋白表达抑制，可引起成骨细胞由G1快速进入S期，致成骨细胞增殖活跃。② p16、p21 基因高甲基化和组蛋白去乙酰化共同介导了其转录和表达抑制，同时DNMT1和HDAC1蛋白表达上调，参与此表观遗传学过程。③ p16、p21 基因组蛋白去乙酰化后分别影响转录因子SP1和STAT1与其相应反应元件的结合，是氟骨症成骨细胞增殖活跃的重要分子机制之一。④ 未观察到 cyclinD1、CDK4基因启动子区甲基化和组蛋白乙酰化参与其表达上调过程。⑤ DNA甲基转移酶抑制剂5-AZA-dC能有效逆转氟所致的p16、p21基因启动子区DNA高甲基化，组蛋白去乙酰化酶抑制剂TSA能有效回升氟所致p16、p21基因转录调控区组蛋白低乙酰化水平，进而回复其转录及蛋白表达，为氟中毒防治提供了新的思路和方向。上述发现从表观遗传学角度阐释了氟骨症发生机制，深化了对氟致病机制的认识，同时表观遗传学改变具有可逆性，使其有望成为氟中毒预防、治疗的新靶点。