ClC3氯通道参与调控糖尿病视网膜新生血管形成及其机制研究

Our previous studies have shown that ClC-3 encodes the volume regulated Cl- channels, ClC-3 chloride channels involved in the regulation of cell proliferation, apoptosis, inflammation, insulin secretion, and other important pathological changes. Recently we have found that there were the volume regulated Cl- channels and ClC-3 expression in retina endothelial cells, high glucose could upregulate the expression of ClC-3 and the current amplitude of volume regulated Cl- channels. After transfection of the ClC-3 siRNA could obviously reverse the upregulation of VEGF induced by high glucose. On the basis, in this study we intended to overexpress or knockdown of ClC-3, to clarify the effect of this channel on cell proliferation, cell permeability and tube formation induced by high glucose, and to reveal the specific mechanism of this process. Moreover, we will further investigate the function role of ClC-3 against the neovascularization of Diabetic Retinopathy using ClC-3 knockout mice. Through this study, we can systematically study the effect and mechanism of the volume regulated Cl- channel ClC-3 in neovascularization in Diabetic Retinopathy, suggesting that ClC-3 may be a novel and potential therapeutic target for Diabetic Retinopathy.

我们前期实验证实ClC-3编码了容积调节性氯通道，ClC-3氯通道参与调控细胞的增殖、凋亡、炎症反应和胰岛素分泌等重要病理变化。预实验结果显示，在视网膜内皮细胞有容积调节性氯通道和ClC-3蛋白表达，高糖可上调ClC-3蛋白的表达和容积调节性氯电流的幅度。siRNA转染抑制ClC-3表达后，高糖诱导的VEGF表达被显著抑制。在此基础上，本项目拟采用ClC-3过表达和ClC-3基因敲除小鼠视网膜内皮细胞，观察上述处理对高糖诱导内皮细胞增殖、内皮细胞通透性及内皮细胞小管形成的调节作用。并进一步采用ClC-3基因敲除小鼠观察在体情况下该通道对DR过程中视网膜新生血管生成的影响。本研究工作将较系统的认识ClC-3容积调节性氯通道在DR新生血管生成中的作用及可能机制，为评估ClC-3容积调节性氯通道是否可作为防治DR的新的靶点提供实验室依据。

糖尿病视网膜病变(diabetic retinopathy，DR)是糖尿病眼病中最严重的并发症，视网膜新生血管形成是DR的中心环节。视网膜微血管内皮细胞损伤是其早期的病理变化。以往研究和预实验结果提示ClC-3容积调节性氯通道参与DR新生血管形成，但具体分子机制目前并不十分明确。本研究通过离体和在体实验观察ClC-3氯通道在DR新生血管形成中的作用及其机制。我们的研究发现在视网膜内皮细胞有容积调节性氯通道和ClC-3蛋白表达，高糖可上调ClC-3蛋白的表达和容积调节性氯电流的幅度。下调ClC-3表达可显著抑制高糖诱导的VEGF表达和小管形成。此外，已有研究表明体内代谢紊乱同时也会诱发炎症反应，在糖尿病情况下机体炎症反应水平升高，参与DR新生血管形成。我们的实验结果也表明在视网膜内皮细胞中，高糖可促进炎症反应相关标志物ICAM-1、VCAM-1表达增加，炎症因子TNF-α、IL-1β分泌增加，单核内皮细胞间粘附增加，NF-κB信号通路激活，而利用氯通道阻断剂和ClC-3 siRNA能逆转上述炎症反应。同时，整体动物实验也表明在糖尿病状态下，敲除ClC-3能逆转视网膜组织中的炎症反应和官腔形成。进一步提示ClC-3氯通道可能通过下调VEGF表达，参与调控内皮细胞炎症反应，进而参与DR新生血管形成。通过该项研究可以为ClC-3氯通道作为防治DR的新的靶点提供实验室依据。