糖尿病视网膜病变中Robo4启动子甲基化对视网膜微血管的作用及机制研究

Robo4 is a cell surface protein which is specifically expressed in endothelial cells and is involved in the regulation of retinal microvascular function during the course of diabetic retinopathy (DR). In the previous research, we found that hyperglycemia could enhance the expression of Robo4 in human retinal endothelial cells (HREC), which induced the dysfunction of HREC. Researches into the transcriptional mechanism of Robo4 gene revealed that hyperglycemia could induce the increase of binding sites between the transcription factor SP1 and the Robo4 gene promoter in HREC, but the deeper regulatory mechanism of the differential binding sites remains unclear. We suspect it is related to the epigenetic modification, and put forward the hypothesis that the promoter hypomethylation of Robo4 can upregulate the binding activity between SP1 and Robo4, which elevates the transcription and expression levels of Robo4 and then promotes the progress of DR. On the basis of previous work, we will use HREC to investigate the methylation status of Robo4 gene promoter region, and explore its functions in the transcriptional regulation of Robo4. Furthermore, the mechanism will also be verified in the diabetic animal model, and the influence of methylation status on retinal microvascular system will be explored during the occurrence and development of DR. These studies would give useful evidences to reveal a new molecular pathogenesis of retinal vascular dysfunction in DR, as well as providing the profound basis and therapeutic way for early DR intervention.

糖尿病视网膜病变（DR）中内皮细胞特异性因子Robo4对视网膜微血管功能的影响日益受到关注。前期研究显示高糖培养的人视网膜血管内皮细胞中Robo4表达升高，可导致细胞透通性、管腔形成等功能异常，进一步探讨Robo4表达的调控机制，发现高糖条件可诱导转录因子SP1与Robo4启动子的结合位点增多，但有关结合位点差异的原因尚不清楚，分析其可能与Robo4的表观遗传学修饰有关。我们推测：高糖环境可导致Robo4启动子区低甲基化，使SP1与Robo4启动子结合作用增强，提高Robo4的转录和表达，进而促进DR的发生发展。本研究拟在细胞和分子水平探讨DR发病中Robo4启动子的甲基化水平，及其对Robo4转录调控的作用机制，并于动物实验中验证该机制对视网膜微血管结构和功能的影响。本课题旨在进一步拓展对DR发病分子机制的认识，为早期干预DR提供新的理论依据和研究方向。

既往研究表明，在糖尿病视网膜病变（DR）的发生发展中，转录因子SP1与Robo4基因启动子的结合活性增强，从而升高Robo4的表达、加速DR的进展。为明确上述改变是否与Robo4表观遗传学的异常修饰相关，本项目在细胞和动物水平检测了Robo4启动子区的甲基化水平，验证了与其相关的调控机制及该机制对视网膜血管系统结构和功能的影响。研究结果显示，在DR发病过程中，Robo4启动子区的甲基化水平降低，同时介导DNA主动去甲基化的TET2表达升高、5mC氧化为5hmC的作用增强、SP1与Robo4的结合活性增强以及Robo4下游ZO-1和occludin的表达下降，视网膜血管出现渗漏、新生血管等结构和功能的异常。通过转染慢病毒和腺相关病毒的方式抑制细胞内和动物视网膜内TET2的表达后，5mC氧化为5hmC的作用降低、SP1与Robo4的结合活性下降、Robo4表达下降、ZO-1和occludin的表达升高，且高糖环境引起的视网膜血管系统结构和功能的异常得到改善。由此得出结论，在DR病程中TET2通过将5mC氧化为5hmC使Robo4基因发生主动去甲基化、活化SP1/Robo4通路、上调Robo4并下调ZO-1和occludin的表达，进而引起视网膜血管系统的异常，抑制TET2的表达可有效下调以上通路从而控制DR的发展进程。本研究结果可为临床早期干预和治疗DR提供新的方向，对DR的临床治疗具有一定的理论指导意义。