糖尿病视网膜病变中NADPH氧化酶4表达的表观遗传学调控机制的研究

糖尿病视网膜病变（DR）是不可逆性致盲眼病之一，发病机制未完全阐明。研究表明表观遗传学修饰与糖尿病"代谢记忆"和基因转录密切相关。Nox4在糖尿病动物多种组织表达上调促进了糖尿病并发症的发生发展，但其表达调控机制尚不清楚。本课题组首次发现了一过性高糖处理视网膜血管内皮细胞(REC)可以诱导Nox4持续性表达上调，此外预实验结果还表明糖尿病小鼠视网膜血管Nox4表达上调同时并伴有乙酰化组蛋白水平升高等表观遗传学改变。本课题拟在前期工作的基础上，应用ChIP和Q-PCR检测高糖处理的REC中Nox4启动子相关的组蛋白乙酰化/甲基化修饰，阐明Nox4表达调控的表观遗传学机制；WB检测DR中组蛋白修饰等表观遗传学改变，采用药物或腺病毒载体技术逆向调控视网膜组蛋白修饰观察其对Nox4表达及DR血管渗漏的影响。为丰富DR致病基因转录的表观遗传学调控机制、发掘DR 治疗新靶点提供理论及实验依据。

Diabetic retinopathy (DR) is a leading cause of blindness and its mechanism remains to be elucidated. Previous studies revealed that epigenetic changes are closely associated with diabetic "metabolic memory" and transcriptional regulation. Nox4 is increased in various tissues of diabetic animals including retinas and leads to tissue oxidative damage;however its regulating mechanism is largely unclear. Our group for the first time demonstrated that transient high glucose exposure could induce persistent Nox4 upregulation in human retinal endothelial cells (HREC). Moreover, we found that upregulation of Nox4 in vasculature of diabetic retinas were accompanied by increased histone acetylation and reduced activity of histone deacetylases (HDAC). Based on our preliminary data, we will utilize chromatin immunoprecipitation (ChIP) and quantative PCR (Q-PCR) to detect acetylation and methylation of histones associated with Nox4 promotor in HREC exposed to transient high glucose in this project. Moreover, adenoviral vector will be constructed to reversely modulate histone modification in Nox4 promotor and its effect on Nox4 expression will be evaluated, which will help to further elucidate epigenetic modulating mechanism of nox4 expression in high-glucose treated HREC. Meanwhile, detailed epigenetic change including histone modification in diabetic mouse retinas will be further evaluated by western-blot analysis and/or ELISA-based assasy. Finally, pharmaceutical approach and adenovirus-mediated gene regulating method will be utilized to investigate whether modulating retinal histone modification will affect retinal Nox4 expression and attenuate vascular leakage in DR. Taken together, overall goal of this project is to fully clarifying epigenetic regulating mechanisms of gene transciption during development of DR and creat the therotical and experimental basis for new therapeutic targets of DR.