基于表观遗传学研究胶原诱导性关节炎大鼠风寒湿、风湿热证型形成机制

Rheumatoid arthritis(RA) ，called as obstinate arthralgia,is difficult to treat.The development of this disease has close relationships with the deficiency of healthy qi and the invasion of exogenous evil.There are more researches about the deficiency of healthy qi while the research about exogenous evil is little.How does exogenous evil contribute to the development of arthralgia?And which role does it play in the common syndrome of arthralgia(cold syndrome and heat syndrome)?All these questions are still not very clear.The project considers collagenⅡ-induced arthritis(CIA),a rat model as human model for RA, and the reasearch targets are degenerative synovium,fibroblast-like synoviocytes(FLS) and peripheral blood mononuclear cells(PBMCs).In order to explore the epigenetic mechanism and expound the role the environmental factors play in different syndromes of RA, the differences of aberrant methylation of DNA,histone modification and expression of micro-RNA are detected between the wind-cold-dampness syndrome and the wind-dampness-heat syndrome,also the epigenetic changes before and after the treatment with Aconite Decoction and Xuanbi Decoction respectively on the same syndrome are detected.In the meanwhile the project observes the promoter methylation level of tumor necrosis factor-α(TNF-α),which is connected closely to RA.By those methods above we want to give new scientific connotation to pathogenesis of Bi syndrome and lay a foundation to research different syndromes of RA objectively and standardized.At the same time the project explore new thoughts to explain the essence of syndrome in TCM more comprehensively.

类风湿关节炎（RA）作为一种治疗棘手的"顽痹"，其发病与正气不足和外邪侵犯关系密切。"正虚" 方面研究较多，"外邪"环节研究不足，外邪以何种方式影响痹证的形成？在形成痹证的最常见的寒、热证型中发挥什么样的作用？这些问题至今不是十分清楚。本项目以大鼠胶原诱导性关节炎为类风湿关节炎模型，病证结合，以病变关节滑膜、成纤维样滑膜细胞、外周血单个核细胞等为研究对象，从表观遗传学角度，比较风寒湿型、风湿热型两种证型之间，以及同一证型分别用乌头汤、宣痹汤治疗前后总DNA甲基化、组蛋白异常修饰、micro-RNA异常表达的变化，并观察与RA发病关系极为密切的肿瘤坏死因子α基因启动子甲基化水平，探讨寒热两种不同证型形成的表观遗传学机制，阐明环境因素在类风湿关节炎不同证型形成中的作用，为中医痹证发病学赋予新的科学内涵，且为RA证型客观化、标准化研究奠定一定基础，为更加全面阐述中医"证"的实证探索新思路

本课题在大鼠胶原诱导性关节炎（CIA）基础上制作风寒湿型关节炎模型（WCD），从表观遗传学角度，探讨“风寒湿外邪”在大鼠关节炎发病中的作用。体外实验表明：风寒湿因素能够使WCD组大鼠关节炎指数评分≥5分的比例升高，表明“风寒湿外邪”可能会加重关节炎严重程度。在体实验表明：与CIA组相比，WCD组外周血单个核细胞（PBMCs）整体基因组DNA甲基化水平显著下降，但两组间PBMCs中DNA甲基转移酶1(DNMT1) mRNA的表达、甲基CpG结合结构域2(MBD2) mRNA的表达、组蛋白H4乙酰化和组蛋白H3-K4、H3-K27的甲基化水平均无明显差异；经乌头汤治疗后DNMT1 mRNA表达和组蛋白H4乙酰化水平接近正常，表明“风寒湿外邪”可能对关节炎模型的大鼠PBMCs部分基因的修饰具有一定作用。本实验建立风寒湿病证结合模型，研究风寒湿因素如何通过表观遗传机制影响类风湿关节炎发病，为中医痹证病因学赋予科学内涵。