TLR4 /ROS信号通路在AMD发病中的作用机制研究

Our team has previously revealed that elevated circulating low-density lipoproteins (LDL) can lead to lipids deposition and RPE dysfunction, which represent for the early age-related macular degeneration (AMD)-like change. But the exact mechanisms are not definitely known. Recent studies suggest that OX-LDL binds to TLR4 and activate the downstream inflammatory pathway to initiate immuno-inflammatory reaction, which is closely related to AMD. So we will set the previous studies as base and do further studies using various biological technologies such as electron microscope、immunofluorescence staining、RT-PCR、western blot、RNAi and translocation to detect: (1) the influence of LDL and oxidized-LDL (oxLDL) to choroid-Bruch-RPE complex of TLR4-/- mice and WT-mice; (2) the gene expression change of RPE cells treated with LDL and oxLDL; (3) the protein expression and phosphorylation level change of TLR4、Lyn、Nox and also ROS and other cytokines production by RPE cells after treated with LDL and oxLDL. Above all, we will elucidate TLR4 / ROS signal pathway in the early onset of AMD mechanism, and thus provide academic basis for further prevention and treatment for AMD.

最近，本课题组发现循环血液中低密度脂蛋白（LDL）增高能引起SD大鼠视网膜发生年龄相关性黄斑变性（AMD）样改变，但作用机制尚不清楚。研究表明，脂质可通过TLR4受体引起的固有免疫介导的炎症通路介导细胞损伤，而免疫炎症反应与AMD发病密切相关。因此本研究拟在原创性研究的基础上，分别在细胞和整体动物水平采用电镜、免疫荧光、RT-PCR、western blot、RNA干扰、转染等技术和方法：（1）观察LDL及OX-LDL对TLR4-/-及WT小鼠视网膜功能及脉络膜-Bruch-RPE复合体的影响；（2）探讨RPE细胞中LDL及OX-LDL对TLR4的表达变化机制；（3）研究LDL及OX-LDL对RPE细胞中TLR4、Lyn蛋白表达、ROS生成和炎症细胞因子表达的影响。通过上述研究，阐明TLR4 /ROS信号通路在早期AMD发病中作用机制，为将来临床早期预防和治疗AMD提供新的靶点。

最近，本课题组发现循环血液中低密度脂蛋白（LDL）增高能引起SD 大鼠视网膜发生年龄相关性黄斑变性（AMD）样改变，但作用机制尚不清楚。研究表明，脂质可通过TLR4 受体引起的固有免疫介导的炎症通路介导细胞损伤，而免疫炎症反应与AMD 发病密切相关。因此本课题在原创性研究的基础上，给TLR4-/-和WT小鼠尾静脉注射LDL，发现在WT小鼠中，ERG振幅明显降低，光感受器细胞功能明显受到影响，而TLR4-/-小鼠电生理未见明显变化 。进一步通过病理切片，比较TLR4-/-和WT小鼠脉络膜-Bruch-色素上皮复合体的整体改变，电镜下测量Bruch膜厚度，观察复合体的超微结构。发现在TLR4-/-中Bruch膜厚度与正常组相比无明显差异，超微结构基本正常。体外予以LDL及OX-LDL环境下培养的RPE细胞，用流式细胞及TUNEL比较观察RPE细胞凋亡情况，数据表明OX-LDL诱导RPE凋亡部分依赖于Erk-Bax/Bcl-2信号通路。课题组成员还发现OX-LDL 上调RPE 细胞中TLR4表达、ROS和炎症细胞因子IL-6分泌增多的影响。通过上述研究，阐明TLR4 /ROS 信号通路在早期AMD 发病中作用机制，为将来临床早期预防和治疗AMD 提供新的靶点。项目研究成果在 Current Eye Research杂志上发表