GRP78调控自噬参与视网膜色素上皮细胞存活机制研究

The dry form of AMD is characterized by starting with progressive injury and death of retinal pigment epithelium (RPE) cells. However, the mechanism of such RPE loss remains complicated and unclear. Our previous research revealed that endoplasmic reticulum (ER) stress mediated apoptosis plays an important role in RPE cell injury and death. And through transmission electron microscopy, we observed the significant change of autophagy level in RPE cells, which closely related to the expression of ER stress chaperone GRP78. Recently, it has been found that autophagy is an important endogenous cellular defense mechanisms. Thus, we suppose that GRP78 is the key molecules to help RPE cells alleviate ER stress by regulating autophagy pathway. On the basis of present findings, this project will adopt dry AMD model to firstly investigate the role of autophagy in RPE cell injury and apoptosis. Secondly, the correlation between GRP78 and autophagy activation will be studied through over-expression or gene interference technology. Finally, knockdown techniques will be used to detect whether GRP78 can regulate RPE autophagy by AMPK/mTOR signal pathway during ER stress. Our research will help to enhance the understanding of RPE cell apoptosis/survival mechanism in dry AMD, which will provide new ideas for drug development.

视网膜色素上皮（RPE）的损伤和死亡是干性年龄相关性黄斑变性（AMD）病理改变的始动环节，其机制复杂且未完全明了。课题组前期通过A2E联合蓝光体外模拟干性AMD，发现内质网应激介导的凋亡途径参与了RPE细胞损伤和死亡过程；同时透射电镜观察到RPE细胞自噬水平异常变化，且与内质网应激分子伴侣GRP78表达趋势相关。新近发现自噬是一种重要的细胞内源性防御机制，推测GRP78通过调控自噬帮助RPE细胞应对内质网应激。以此为基础，本项目采用现有体内外干性AMD模型，首先通过双向调节自噬，明确其在内质网应激介导的RPE细胞凋亡中作用；其次利用过表达/基因沉默技术，研究GRP78与自噬以及RPE细胞存活的相关性；最后通过分步基因干扰检测GRP78/AMPK/mTOR信号通路表达，阐明GRP78调控自噬的分子机制。课题的完成将完善干性AMD中RPE细胞凋亡/存活的分子机制，也为药物研发提供新思路。

视网膜色素上皮（RPE）在维持视网膜稳态和视功能中起着至关重要的作用。年龄相关性脂褐素A2E的积聚并在蓝光诱导下致RPE细胞损伤是视网膜退行性疾病共同的始动环节。课题组前期研究发现，内质网应激参与了A2E及蓝光诱导的RPE细胞损伤过程，同时透射电镜观察到细胞内自噬水平变化。本课题研究中通过Western blot、免疫荧光染色等方法发现RPE细胞损伤后自噬水平升高。进一步，运用慢病毒干扰内质网应激分子伴侣GRP78表达，发现AMPK磷酸化水平降低，同时磷酸化mTOR活性增强，从而下调了RPE细胞自噬水平。此外，抑制自噬增加了A2E及蓝光诱导的RPE损伤。相反，自噬激动剂雷帕霉素能减轻内质网应激相关凋亡蛋白CHOP和caspase-12表达，一定程度上保护RPE细胞。本课题初步揭示了GRP78通过AMPK/mTOR通路调控自噬是RPE细胞重要的内源性保护机制之一。课题的的完成将完善视网膜退行性疾病中RPE细胞凋亡/存活的分子机制，进而为视功能保护提供理论依据。