apoA1抑制HIF-1a/VEGF通路抵抗视网膜新生血管形成的分子机制研究

Proliferative diabetic retinopathy (PDR)，the retinal neovascularization stage, resulted in blindness of a large number of diabetic patients. We previously confirmed that there were decreased serum apolipoprotein A1 (apoA1) levels in PDR patients, and apoA1 was expressed in human retina, suggesting apoA1 might play a role in the retina. In preliminary experiments, we found that apoA1 could promote tight junction protein expression, and decrease VEGF concentrations of cells. As reported, apoA1 mimetic peptide inhibited tumor angiogenesis by inhibiting HIF-1а and VEGF levels, and PI3K/Akt and MEK/ERK were the most important signaling pathways of affecting HIF-1а. Our study intended to build the retinal neovascularization animal model of apoA1 knockout mice, APOA1 transgenic mice and wild-type mice, and cell in high glucose, combined with the biological sample testing. For the first time we would verify that apoA1 would inhibit PI3K and/or ERK1/2 pathway, then decrease HIF-1а/VEGF level, finally resist retinal neovascularization from three levels of whole, cell and tissue (proliferative membranes). This study would support theoretically that the injection of safe and effective apoA1 complex could resist retinal neovascularization.

增殖性糖尿病视网膜病变（PDR）-视网膜新生血管阶段，可导致大量患者失明。我们前期证实：PDR患者血清载脂蛋白A1（apoA1）水平降低；apoA1在人视网膜各层均表达，提示apoA1可能在视网膜中起作用。预实验中我们发现：apoA1促进视网膜紧密连接蛋白表达，并降低细胞VEGF浓度。已知apoA1模拟肽通过抑制HIF-1а及VEGF而抑制肿瘤新生血管；PI3K/Akt和MEK/ERK是影响HIF-1а的最重要信号通路。本项目拟构建apoA1敲除鼠、APOA1转基因鼠和野生鼠的视网膜新生血管动物模型及高糖细胞模型，结合生物样本检测，首次从整体-细胞-组织(增殖膜)三层次验证“apoA1通过抑制P13K/Akt和（或）MEK/ERK通路下调HIF-1a/VEGF，抵抗视网膜新生血管形成”这一假说，为人体注射安全及有效的apoA1复合物进入抵抗视网膜新生血管形成这一崭新治疗领域提供理论依据。

增殖性糖尿病视网膜病变（PDR）-视网膜新生血管阶段，可导致大量患者失明。我们前期证实：PDR患者血清载脂蛋白A1（apoA1）水平降低；apoA1在人视网膜各层均表达；apoA1促进视网膜紧密连接蛋白表达，并降低细胞VEGF浓度。已知PI3K/Akt和MEK/ERK是影响HIF-1а的最重要信号通路。本研究中，我们成功构建apoA1敲除鼠、APOA1转基因鼠和野生鼠的视网膜新生血管动物模型及高糖细胞模型，通过PI3K抑制剂或MEK1/2抑制剂处理，并结合apoA1模拟肽D-4F体内体外实验多层次进行验证“apoA1通过抑制P13K/Akt和（或）MEK/ERK通路下调HIF-1a/VEGF，抵抗视网膜新生血管形成”这一假说。 . 重要实验结果总结：1.体内实验：利用视网膜成像系统及视网膜铺片等发现三种小鼠OIR模型中，主要动脉、静脉迂曲扩张程度无明显差异；无灌注区面积大小、血管扭曲程度比较结果为：apoA1-/-小鼠OIR模型>正常小鼠OIR模型>APOA1转基因小鼠OIR模型；在OIR模型中，腹腔下注射PI3K抑制剂组，小鼠视网膜毛细血管网较腹腔下注射MEK1/2抑制剂组密集，无灌注区面积减少，提示此新生血管形成的过程中，PI3K通路可能起主导作用；腹腔注射30mg D-4F组视网膜主要血管迂曲程度比D-4F 10mg组减少，片状出血灶比10mg组减少。2.体外实验：利用腺相关病毒载体及RNAi技术沉默技术，成功构建apoA1在体外高糖培养下HRMECs高低表达，发现与未干预组相比，PI3K抑制剂组HRMECs增殖能力明显降低，并随浓度升高增殖能力进一步降低；在细胞高糖环境中，发现随着D-4F浓度增加，akt及p-akt蛋白表达水平均增高。 . 综上实验结果我们得出结论:1.apoA1基因表达量的多少对视网膜主要动脉、静脉无明显差异；2.apoA1基因高表达抑制新生血管形成，保护视网膜屏障；apoA1基因敲除后可促进新生血管形成；3.在新生血管形成过程中，PI3K/Akt通路可能起主导作用；4.apoA1模拟肽D-4F 可能具有保护视网膜屏障的作用。. 本研究为人体注射安全及有效的apoA1复合物进入抵抗视网膜新生血管形成这一崭新治疗领域提供理论依据。