叶酸对动脉粥样硬化表观遗传学作用机制的研究

The aim of this study is to explore the epigenetic effects of folic acid on atherosclerosis (AS) by DNA methylation. The relationship between folic acid, DNA methylation and AS will be studied by the three experimental levels and three approach experiments. The three experimental levels are human umbilical vein endothelial cells (HUVEC) model, AS animal models and patients with coronary heart disease. Case-control study will be conducted in the patients with coronary heart disease. The three approach experiments are the methyl metabolism, DNA methyltransferase (DNMT) activity and expression levels, the changes of DNA methylation in HUVEC and animal models, patients with coronary heart disease as well as controls. The products of methionine circulation, the activities and expressions of DNMT and the levels of methyl donor will be detected to explore the effects of folic acid on DNA methylation metabolic process in AS. The genome-wide methylation status will be detected by using Methylated DNA Immunoprecipitation sequencing (MeDIP-seq) assays to explore the molecular mechanism of folic acid on DNA methylation in AS. The diverse DNA methylation maps of AS cell and animal models, patients with coronary heart disease as well as controls will be constructed by MeDIP-seq technology. The spectrum of genomic methylation changes will be compare in AS cell and animal models, patients with coronary heart disease as well as controls by bioinformaitcs. The differentially DNA methylated regions from AS cell and animal models, patients with coronary heart disease as well as controls will be identified and their functions will be studied. The effect mechanism of folic acid on the genes of DNA methylation in AS will be explored. The research results will provide epigenetic information to explore the molecular mechanisms in AS development and provide scientific evidence for exploring molecular markers in AS diagnosis, treatment and prognosis. The objective of the research is to develop new approach of prevention and treatment of AS.

本研究基于国内外研究现状、相关理论及我们前期研究基础，采用血管内皮细胞氧化损伤模型、大鼠动脉粥样硬化模型及冠心病患者病例对照研究，确定叶酸-DNA甲基化-动脉粥样硬化（atherosclerosis，AS）三者之间的关联。通过检测叶酸对血管内皮细胞氧化损伤模型、大鼠动脉粥样硬化模型及冠心病患者和对照组的蛋氨酸循环代谢产物、甲基转移酶活性和表达水平、甲基供体水平，明确叶酸对AS甲基化代谢途径的作用。采用MeDIP-seq 高通量测序技术，检测血管内皮细胞氧化损伤模型、大鼠动脉粥样硬化模型及冠心病患者和对照组全基因组的甲基化状态，比较其基因组甲基化谱的变化，找出叶酸通过改变哪些基因的甲基化状态作用AS，提出基于DNA甲基化途径叶酸在AS发病中的作用机制。为探讨AS发生发展的分子机制提供表观遗传信息，寻找AS的诊断、治疗或预后相关分子标志物提供依据，为AS的防治开辟新的途径。

动脉粥样硬化（AS）是一种多因素相关的疾病，表观遗传学改变可能是AS 发生发展的重要病理学机制之一。叶酸参与一碳单位的代谢与DNA甲基化的作用。本研究基于国内外研究现状、相关理论及我们前期研究基础，采用血管内皮细胞氧化损伤模型、动脉粥样硬化动物模型及冠心病患者病例对照研究，确定叶酸-DNA甲基化-AS三者之间的关联。体外研究采用氧化低密度脂蛋白（ox-LDL）和同型半胱氨酸（Hcy）干预人脐静脉内皮细胞（HUVECs）两种氧化损伤模型；体内研究采用Wistar大鼠模型和载脂蛋白E基因敲除（apoE-/-）小鼠两种动物模型，采用Pyromark短序测序甲基化分析技术。研究结果显示，叶酸可以通过提高细胞甲基化潜能和调节甲基转移酶活性，增强机体的抗氧化能力和降低炎症反应，改善动脉粥样硬化；叶酸可改变基因组甲基化谱，使单核细胞趋化蛋白-1（MCP-1）和血管内皮细胞生长因子（VEGF）基因甲基化并静默，进而抑制动脉粥样硬化相关蛋白对血管内皮细胞的有害作用，减少细胞凋亡水平。病例对照研究收集了正常冠脉对照组、AS组和冠心病（CAD）三组，检测了蛋氨酸循环代谢产物、甲基供体水平、甲基转移酶活性和表达水平，采用Illumina 850K甲基化芯片技术，检测了冠心病患者和对照组全基因组的甲基化状态，比较其基因组甲基化谱的变化。结果显示，血浆中S-腺苷同型半胱氨酸（SAH）浓度随着冠脉狭窄程度加重呈明显上升趋势，且在冠脉造影人群中，血浆SAH与冠脉病变独立相关，SAH可能是冠心病早期诊断的一个新型生物标记物。血清叶酸及甲基化指数S-腺苷甲硫氨酸（SAM）/SAH是冠心病的保护因素，而血清SAH是冠心病的独立危险因素；叶酸水平可调节人类巨噬细胞脂质负荷和炎症相关孤核蛋白受体NR4A2甲基化水平，并调节外周血中NR4A2的基因表达，NR4A2可调节下游TNF-α，MCP-1及VEGF的mRNA 表达，从而抑制动脉粥样硬化。该研究结果为探讨叶酸通过DNA甲基化途径防治AS的作用机制提供依据，为AS的防治开辟新的途径。