SIRT1介导的表观遗传学修饰在氟中毒卵巢功能损伤中的调控作用及机制研究

The molecular mechanism of ovarian function damage with fluorosis is unknown. And the effective measures to block the damage by fluoride are also lack. It has shown by the preliminary experimental results that DNA methylation and SIRT1 were associated with ovarian function damage with fluorosis. Therefore, we adopt the research methods such as epigenetic research means to explore the epigenetic regulation mechanism of ovarian function damage with fluorosis and the measures of SIRT1 intervention. The main research contents include: (1)We will identify the epigenetic modification features which related with ovarian function damage with fluorosis using the human DNA methylation chip, miRNA expression profile chip and multiple omics data integration analysis; (2)We will verify the selected DNA methylation and the miRNA sites which regulate the ovarian granulosa cell apoptosis using the animal model with fluorosis; (3) We will evaluate the intervention effect to block the cell apoptosis using human ovarian granulosa cells induced by fluoride transfected with SIRT1 activator and inhibitor. We focus on the molecular mechanism for the new hypothesis of "fluoride - abnormal epigenetic regulation- the abnormal expression of gene and protein related with ovarian function damage". We want to make clear the gene expression patterns and characteristics of DNA methylation and miRNA in the ovarian granulosa cells induced by fluoride. We want to clarify the epigenetic mechanisms of ovarian function damage with fluorosis and provide the new target point for the new method of prevention and control ovarian function damage with fluorosis.

氟中毒卵巢功能损伤分子机制不明，缺乏有效阻断措施。前期研究提示DNA甲基化和SIRT1可能与氟中毒卵巢功能损伤有关，因此拟采用表观遗传学等研究手段深入探讨氟中毒卵巢功能损伤的表观遗传调控机制，探索SIRT1干预防治措施。内容包括：(1)采用人DNA甲基化芯片、miRNA表达谱芯片及多组学数据整合分析鉴定与氟中毒卵巢功能损伤密切相关的表观遗传学修饰特征；(2)采用动物实验验证筛选的DNA甲基化和miRNA位点参与调控氟中毒卵巢颗粒细胞凋亡；(3)采用SIRT1激活剂和抑制剂转染人卵巢颗粒细胞，评估SIRT1干预阻断氟诱导卵巢颗粒细胞凋亡的效果。项目集中研究新的“氟-表观遗传调控异常-卵巢功能损伤相关基因、蛋白表达异常”分子机制假说，揭示氟中毒卵巢颗粒细胞DNA甲基化和miRNA参与基因表达调控的作用模式和特征，为深入研究氟中毒卵巢功能损伤的表观遗传作用机制提供参考，为研发防治新方法提供靶点。

采集了氟中毒和正常对照组不孕女性取卵后的卵泡液，梯度离心法收集卵巢颗粒细胞，TRizol法提取RNA和DNA进行了DNA甲基化测序和miRNA测序，获得了氟中毒女性miRNA和DNA甲基化表达谱，通过多组学数据整合分析确定了与卵巢功能损伤密切相关的DNA甲基化ATG12和miR-378d位点，对差异表达miR-378d的实时定量PCR验证，和ATG12的甲基化PCR和实时定量PCR结果证实了测序结果；采用饮水添加不同浓度氟化钠（0mg/L、50mg/L、100mg/L和150mg/L）的雌鼠模型，分别在染毒60、120和180天，采集氟中毒雌鼠和正常对照组雌鼠血液、卵巢、子宫、肝肾等，提取血液RNA、DNA和蛋白，实时定量PCR等实验验证了筛选的ATG12甲基化和miR-378d位点在氟中毒雌鼠卵巢颗粒细胞凋亡和自噬的调控作用；采用不同浓度SIRT1激活剂（白藜芦醇）和抑制剂（烟酰胺）处理人卵巢颗粒细胞系，流式细胞术检测细胞凋亡，结果发现10、20、30和40µM白藜芦醇抑制细胞凋亡，而1、5、10和15mM烟酰胺促进细胞凋亡，SIRT1干预可阻断氟诱导的卵巢颗粒细胞凋亡。研究结果表明miRNA参与氟中毒卵巢功能损伤，其中miR-378d可能是关键分子，而细胞自噬可能也起重要作用，在氟化钠浓度较低（1x10-3和2x10-3mM NaF）时先增加细胞自噬以保护细胞，随氟化钠浓度的增加，细胞凋亡的作用占据主导，抑制细胞自噬，从而导致卵巢颗粒细胞凋亡，雌激素分泌减少，导致卵巢功能损伤。本研究结果表明通过对细胞自噬和细胞凋亡的调控可能阻断氟化钠的毒性作用，其中miR-378d和SIRT1相关分子药物可能会对氟中毒女性的生殖功能起到调控作用，抑制氟中毒的卵巢毒性。本研究成果为氟中毒卵巢功能损伤机制研究提供了科学依据，其结果对氟中毒女性生殖功能损伤防治具有一定的临床应用价值。