同型半胱氨酸经Rap1A调控RIG-I/MDA5通路介导免疫炎症反应表观遗传机制研究

Immune-Inflammatory reactions run through the whole pathogenic processes of Homocysteine (Hcy), and Rap1A is an important gene which acts as a “molecular switch” in down-stream signal transduction pathways. RIG-I/MDA5 pathway plays a vital role in the activation and amplification of immune inflammation, and Histone and DNA methylation are important mechanism of Hcy pathogenesis, but whether and how Hcy regulate RIG-I/MDA5 signaling pathway to lead to inflammatory immune response via Rap1A is still not clear. Therefore, Confocal Laser Scanning Microscope will be used to engage in the qualitative and quantitative analysis of Rap1A, so as to explicit the effect of which for Hcy-induced inflammatory immune response. To construct 3’UTR vector of MDA5 and RIG-1 and co-transfection with RapA1-Luc reporter gene, and take the measures of intervention from different levels such as Rap1A, RIG-1 and MDA5 respectively, then analyse the changes of IL-1 and TNF-α, for the sake of revealing that RIG-I/MDA5 signaling pathway mediated by Rapa1 is an important mechanism in Hcy-induced immune inflammation. Using the blocking strategy based on the bidirectional regulation of Histone and DNA methylation to explore the influence on the target gene after their balance is broken respectively, and reveal the mechanisms of interaction between them which modulate Rap1A to cause immune inflammation response, to provide the experimental basis for the prevention and treatment of Hcy-induced disease.

免疫炎症反应贯穿于同型半胱氨酸(Hcy)致病的全过程，Rap1A是下游通路中起“分子开关”作用的重要基因，而RIG-I/MDA5通路是炎症活化与放大的关键通路，且组蛋白和DNA甲基化是Hcy致病的重要机制，但Hcy是否通过Rap1A调控RIG-I/MDA5通路引起免疫炎症反应尚未清楚。因此拟采用共聚焦显微镜等观察Rap1A的变化并定位，确定其是Hcy引起免疫炎症反应的重要基因；构建MDA5和RIG-1启动子区3’非编码序列载体共转染RapA1-Luc报告基因，分别从Rap1A、RIG-1和MDA5等层面干预后，分析IL-1和TNF-α等变化，阐明Rap1A调控RIG-I/MDA5通路在Hcy引起免疫炎症反应中的作用；采用阻断为主的策略从组蛋白和DNA甲基化双向调控入手，探讨各自平衡模式被打破后对靶基因的影响，揭示两者相互作用调控Rap1A引起免疫炎症反应的机制，为防治Hcy提供实验依据。

景：免疫炎症反应贯穿于同型半胱氨酸(Hcy)致病的全过程，Rap1A是下游通路中起“分子开关”作用的重要基因，DNA甲基化和转录因子的激活是Hcy致病的重要机制，但Hcy是如何调控Rap1A的表达引起免疫炎症反应尚未清楚。.内容：采用多种方法检测Rap1A的变化，确定Rap1A是Hcy引起免疫炎症反应的重要基因；构建FOXO1和DNMT1干扰/过表达载体共转染巨噬细胞，分别从Rap1A、FOXO1和DNMT3A等层面干预后，分析IL-6、TNF-α、M1表型等变化，阐明DNA甲基化和转录因子FOXO1相互作用共同调控Rap1A的表达在Hcy引起免疫炎症反应中发挥了重要作用；从DNA甲基化和转录因子双向调控入手，探讨各自平衡模式被打破后对靶基因的影响，揭示两者相互作用调控Rap1A引起免疫炎症反应的机制。.结果：（1）Hcy作用巨噬细胞后，细胞增殖和迁移能力明显增强；炎症因子IL-6、TNF-α水平增加；细胞趋于M1极化；同时Hcy可以促进Rap1A的表达。（2）干扰Rap1A表达后，细胞增殖和迁移能力下降；M1极化水平降低；炎症因子表达降低。（3）过表达DNMT3A/干扰FOXO1表达使炎症水平降低，Rap1A表达降低，且二者有协同作用。.科学意义：本研究从DNA甲基化和转录因子调控Rap1A入手，补充和完善了Hcy致巨噬细胞炎症的机制，为临床高Hcy血症相关疾病的防治提供理论依据。