CKD病人外周血单核细胞DNA甲基化作用机制及中医虚实证型对其影响的研究

Little is known regarding the relationship between blood monocyte and chronic kidney disease (CKD). Our recent researches discovered that the transcript levels of several genes encoding the enzymes that regulate the DNA methylation were apparently regulated in CKD patients. Some literatures uncoverd the epigenetic regulation of monocyte-to-macrophage differentiation distinguish tolerant and trained macrophage phenotyes in tissues. Therefore, we propose a hypothesis: environmental factors, summarized as “syndrome” in thaditional chinese medcine, imprint some immunological markers in monocyte, which determine the funcitonal fate of monocytes and monocyte-derived macrophages in kidney tissues of CKD patients. In order to verify the hypothesis, we will analyze DNA methylation pattern and screen out the differentially methylated promoters (DMP) of genes in normal and different syndrome CKD patient blood monocyte. Meanwhile, bone marrow-derived macrophage of C57BL/6 mice that regulated by methylation are adoptively transferred into renal ischemia reperfusion injury mice. Inflammational response and fibrosis are researched in the model, which will benefit to verify functions and illuminate mechinisms about DNA methylation. Obviously, these will provide new orientation for diagnosis and treat of CKD.

外周血单核细胞特征是否与CKD病情进展相关尚不清楚。我们前期研究结果提示CKD病人外周血单核细胞表达了明显不同的DNA甲基化相关酶，结合文献报道：具有不同表观遗传印迹的外周血单核细胞可影响渗入组织巨噬细胞的表型及功能。我们据此提出假说：环境因素（中医概括为“证”）通过影响CKD病人外周血单核细胞DNA甲基化而形成免疫印迹，进而影响肾组织单核巨噬细胞的促炎或促纤维化功能，影响CKD病情。为验证本假说，分析炎症反应和纤维化过程的CKD病人及不同中医证型的CKD病人外周血单核细胞DNA甲基化模式，筛选差异甲基化基因，并通过甲基化调节的 C57BL/6小鼠骨髓源性巨噬细胞干预 C57BL/6小鼠肾缺血再灌注损伤模型，观察早期炎症反应及晚期纤维化形成过程，进行功能验证及机制研究，为CKD中西医诊疗及预后判断提供全新思路。

慢性肾脏病（Chronic kidney disease, CKD）的炎症反应及纤维化形成与肾组织不同表型及功能特点的巨噬细胞密切相关，人外周血单核细胞可渗入肾组织形成巨噬细胞，而具有不同表观遗传印迹的外周血单核细胞是否影响肾脏病的炎症反应及纤维化病情尚不明确。中医药防治CKD的辩证分型也亟需标准化。为此，本项目研究了：CKD病人外周血单核细胞DNA甲基化状态及与CKD病情的相关性；DNA甲基化调整的单核细胞对肾脏病炎症反应及纤维化形成的影响及机制；分析了特定中医证型（脾肾气虚及脾肾气虚兼湿热）的CKD病人外周血单核细胞DNA甲基化，筛选上述中医证型的DNA甲基化物质基础（基于“表观遗传学的整体环境因素影响基因表达与中医药的整体观念”相似性）。结果发现：CKD病人外周血单核细胞存在明显的DNA甲基化改变基因，功能及通路预测与其免疫调节功能密切相关，而且CKD早期炎症反应为主患者及CKD晚期纤维化为主患者的外周血单核细胞也表达明显不同的DNA甲基化差异基因，功能等预测分析也提示与其功能相关。而DNA甲基转移酶抑制剂及DNA甲基转移酶抑制剂诱导的单核细胞研究提示：低剂量的DNA甲基转移酶抑制剂及DNA甲基转移酶抑制剂诱导的单核细胞可以减低早期肾脏损伤。机制研究提示，DNA甲基转移酶抑制剂可提高肾损伤组织免疫调节性M2型单核巨噬细胞表达及促进单核细胞过氧化物酶体增殖物激活受体γ（PPARγ）表达。而基于DNA甲基化的脾肾气虚兼湿热证型研究提示JAKMIP1、COL6A2等基因的DNA甲基化改变可能作为上述中医证型的微观物质基础。本项目可为CKD诊疗提供全新思路，也提供了客观依据，同时也一定程度上为中医证型的现代化研究指出了一条新方向，可为中医药论治CKD提供切实可行的理论依据。