原发性高血压表观遗传学机制的初步探讨:ABCG4基因启动子DNA甲基化异常在高血压血管重塑中作用及其分子基础

Epigenetic variations may underlie hypertension and in fact, better than variation in DNA sequence, may explain the late onset and quantitative nature of this disease. Genome-wide DNA methylation patterns in hypertensive Dahl S (SS) and normotensive Brown Norway (BN) rats were assessed in a high throughput manner by methylated DNA immunoprecipitation combined with a competitive hybridization on a microarray (NimbleGene) covering 22K promoters. More than 2K promoters were hypermethylated in the SS kidney. The heart methylomes were less different. Gene encoding enzymes and involved in inflammation were the mostly methylated in the hypertensive kidney whereas the genes involved in signal transmission were the least affected. We then zoomed in on the known hypertension QTL areas and further narrowed down hypermethylated targets. We then selected a few candidate genes for a detailed nucleootide specific quantitative methylation assay using pyrosequencing of bisulfite converted DNA. Pyrosequencing revealed hypermethylation of the renin promoter (but not a distal CpG island) in the hypertensive kidney, consistent with the reduced renin expression in this strain. Thus, an epigenetic mechanism may underlie some forms of hypertension. However, since the kidney is both the culprit and the victim of hypertension, the cause-effect relationship between the epigenome and hypertension has to be established.

原发性高血压发病机制非常复杂，迄今尚未阐明。本课题试图从表观遗传学角度探讨DNA甲基化异常与高血压病血管重塑之间的关系。我们在前期实验中通过全基因组DNA甲基化芯片发现自发性高血压大鼠动脉血管ABCG4基因启动子区域较正常对照显著低甲基化，且其mRNA水平和蛋白均高表达。据此，我们认为ABCG4基因的表观遗传学异常参与了高血压血管重构。为进一步探讨ABCG4在血管重塑和血压调节异常中的作用，本项目拟通过慢病毒过表达或RNA干扰抑制ABCG4基因，观察其对血管内皮细胞一氧化氮、炎症因子表达的影响；对主动脉平滑肌细胞增殖、迁移等功能及RAAS系统、氧化应激等方面影响；并采用ABCG4-shRNA尾静脉注射抑制自发性高血压大鼠的ABCG4基因的表达，观察其对血压、RAAS系统、血管炎症因子等指标的影响，从细胞到动物水平确认ABCG4基因甲基化异常与高血压的关系，为临床防治提供新的理论依

原发性高血压发病机制非常复杂，迄今尚未阐明。本课题在前期实验中通过全基因组DNA甲基化芯片发现自发性高血压大鼠动脉血管ABCG4基因启动子区域较正常对照显著低甲基化，且其mRNA水平和蛋白均高表达。据此，我们认为ABCG4基因的表观遗传学异常参与了高血压血管重构。. 在原有的基础上对原发性高血压相关基因的DNA甲基化状态进行全面、细致的分析，已完成自发性高血压大鼠（SHR）和正常对照大鼠（WKY）全基因组DNA甲基化的测序结果，MeDIP-Seq 测序结果结合mRNA表达谱进行联合分析18个DNA甲基化的低mRNA表达以及12个DNA低甲基化的高mRNA表达结果和比例。. 为进一步探讨ABCG4在血管重塑和血压调节异常中的作用，本项目拟通过RNA干扰抑制ABCG4基因，观察其大鼠平滑肌细胞的影响，从细胞、蛋白、基因水平各方面阐明ABCG4基因抑制对大鼠平滑肌细胞功能的影响。ABCG4可以通过改变p67phox的表达，影响细胞内的活性氧的水平。ABCG4还可以改变和AT1R，eNOS，NF-KB和VEGF的水平，提示其可能在血管重构中发挥了重要的作用。确认ABCG4基因甲基化异常与高血压的关系，为临床防治提供新的理论依据。