冠心病痰湿体质调治的表观遗传学调控机制研究

The persons of phlegm-dampness constitutions are high risk group of Coronary heart disease.The special status of TCM constitution is formed on the difference of multiple gene expression levels.At present, the unique gene expression profile of phlegm-dampness constitution have been discovered by gene chip technique. But from the perspective of gene polymorphism, it conuldn't explain the conflict between the fixed gene sequence and the viewpoint of adjustable constitution.So far only found a small amount of associated gene polymorphisms (SNPs), in contradiction with the phenomenon of the actual existence of a large number of differences in the expression of genes. Epigenetic regulation does not change the gene sequence,it affect gene expression through the modification of genes and chromosomes.The reversibility characteristics of epigenetic regulation provides a new way for the mechnism of the viewpoint of adjustable TCM constitution.Recently, it was reported in many studies that altofrequent abnormal methylation of the CpG islands in the gene promoter region occurred on the occurrence and development of atherosclerosis, aberrant DNA methylation is a common phenomenon in the pathologic process of atherosclerosis.Compared with the peaceful quality, phelegm-dampness constitution, Our previous studies found that there were the aberrant DNA methylation in the gene promoter region in 47 kinds of genes.This research is going to use the coupling technique of methylated DNA immunoprecipitation and DNA methylation chip to find out the specific DNA methylation pattern of the CHD patients with hlegm-dampness constitution, to use the integrated TCM modulating interventions to improve the phlegm-dampness constitution and evaluating its effect, to analysis the differences of DNA methylation profile between the before and after intervention,to provide the epigentic basis of the mechanism of TCM constitution adjustable view.

痰湿体质是冠心病的易感体质，体质差异是多基因表达水平差异的外在特征反映，已发现痰湿体质具有独特的基因表达谱。多从基因多态性角度分析不同体质基因表达差异机制，但基因多态性的碱基顺序终生不变，与中医体质可调论相矛盾，且目前仅发现少量相关联的基因多态性位点，与实际存在的大量差异表达基因相矛盾。表观遗传学调控不改变基因序列，通过基因和染色体的修饰影响基因的表达，具有可逆性,为中医体质可调论的机理研究提供了新的思路。在动脉粥样硬化病变中存在高频率的基因CpG 岛异常甲基化事件，前期研究发现与平和质相比，痰湿体质存在47种甲基化差异基因。本课题拟联用甲基化DNA免疫共沉淀与DNA甲基化芯片技术，快速高效地发现冠心病人痰湿体质独特的甲基化差异相关基因，采用综合调治措施改善痰湿体质，继而比较冠心病痰湿体质改善前后可能出现的DNA甲基化谱差异及其与体质干预效果之间的关系，为中医体质可调论提供表观遗传学依据。

痰湿体质是冠心病的易感体质，体质差异是多基因表达水平差异的外在特征反映，已发现痰湿体质具有独特的基因表达谱，但仅发现少量相关联的基因多态性位点，与实际存在的大量差异表达基因相矛盾，表观遗传学调控理论为中医体质可调论的机理研究提供了新的思路。本项目采取Illumina Human Methylation 450K Beadchip全基因组甲基化芯片检测平和质和痰湿质者血液全基因组48万个甲基化位点甲基化水平，并进行聚类分析、主成分分析、GO功能注释和KEGG信号通路分析，结果发现与平和质相比，痰湿质者有2766个探针的甲基化程度差异显著（P<0.01），其中甲基化程度增高者有1601个，甲基化程度下降者有1165个，涉及2217个基因，KEGG通路分析发现ECM受体作用信号通路、粘着斑、Hippo信号通路等被显著富集。与平和质相比，痰湿质组甲基化差异显著的心血管相关基因有44个基因（50个甲基化探针位点），其中甲基化程度增高的心血管相关基因28个（33个探针），甲基化水平更低的基因13个（17个探针），另外IRS2、PON3和LRP12等3个基因有多个甲基化差异不一致的探针位点。针对上述基因采用Methylight法进一步检测痰湿体质群调治前后上述基因启动子区的甲基化水平，结果发现呈现显著性差异的7种高甲基化基因和4种低甲基化基。动物层面研究发现药物、运动和饮食调控都可显示改善动物的血脂水平和心肌损伤程度，但其中单纯药物调控效果最差，综合调控组和行为方式组的低密度脂蛋白受体、低密度脂蛋白受体相关蛋白1和低密度脂蛋白受体相关蛋白12呈现基因启动子区甲基化增高的现象，而氧化低密度脂蛋白受体1出现甲基化水平降低的现象。本研究发现对于研究中医体质的实质和基因诊断，对于指导临床个体化用药和"治未病"具有现实意义。