早期AMD视网膜Aβ毒性作用与血视网膜外屏障P-糖蛋白关系的研究

The pathogenesis of age-related macular degeneration (AMD) remains unclear and no effective treatment available. It is increasingly clear that the deposits of amyloid beta possibly contribute to the damage of retina in the early stage of AMD. We have reported that signaling pathways of p-glycoprotein located in the outer blood-retinal barrier involved in diabetic retinopathy in vitro. Recently,in preliminary experiments, we found that amyloid bata in low concentration can inhibit the expression and activity of p-glycoprotein both in fresh retinal pigment endothelium cells from na?ve mice and human retinal pigment endothelium cell line D407. Therefore, based on the amyloid beta transport clearance theory of Alzheimer's disease, we now speculate that p-glycoprotein in the outer blood-retinal barrier is a therapeutical target for reducing amyloid beta deposit and attenuating its neurotoxicity in the retina of early AMD. First, using our established in vitro blood-retinal barrier models, we will carry on our investigation in regulatory mechanism of p-glycoprotein mediated amyloid beta increasing and neurotoxicity. Second, we will adjust the p-glycoprotein signaling pathway to assess the role of p-glycoprotein in na?ve mice with intravitreous injection of amyloid beta in low concentrations. Finally, using Tg2576 transgenic mouse model of human cerebral amyloid pathology, we will exam the roles that p-glycoprotein signaling pathway plays in the increasing amyloid beta levels and inducing damage in electroretinogram and apoptosis in the retina. This project will potentially tell us whether p-glycoprotein in the outer blood-retinal barrier is involved in neurotoxicity of amyloid beta, which may eventually lead to the clarification of p-glycoprotein mediated amyloid beta mechanism in the early stage of AMD.

年龄相关性黄斑变性（AMD）发病机制仍不清楚，而早期AMD视网膜β淀粉样蛋白（Aβ）增高和损害的证据日益增多。已报道血视网膜外屏障P-糖蛋白信号介导机制，初步发现低浓度Aβ可抑制视网膜色素上皮细胞P-糖蛋白表达与活性。借鉴阿尔茨海默氏病的Aβ转运清除学说，推测血视网膜外屏障P-糖蛋白是早期AMD视网膜Aβ增高与损伤的干预靶点。拟继续采用已有血视网膜外屏障模型，完善Aβ抑制P-糖蛋白表达与活性的研究；随后，通过活化和抑制P-糖蛋白信号通路，观察低浓度Aβ玻璃腔注射对小鼠视网膜Aβ含量、外排、视网膜电图和凋亡等的影响；最后采用阿尔茨海默氏病转基因小鼠模型，调节P-糖蛋白信号通路，全面观察视网膜Aβ外排、视网膜电图和凋亡等的改变，进一步验证假说。可望初步明确血视网膜外屏障P-糖蛋白信号通路是否参与早期AMD视网膜的Aβ增高与损伤。

年龄相关性黄斑变性（AMD）是目前老年人群中不可逆性视觉损害的致盲首因。随着人口老龄化进程加快和医学诊疗水平提高，AMD发病率在我国呈现逐年增高的趋势。然而，截至目前AMD发病机制仍未完全阐明，亦无有效治疗手段。近年来，β-淀粉样蛋白（Aβ）细胞外沉积导致的Aβ级联反应学说被认为可能是AMD病理特征之一，视网膜Aβ增高、聚集形成的玻璃膜疣也被认为参与了早期AMD的视网膜损伤。血视网膜屏障负责视网膜营养物质的进入和废物的排出，其转运功能的异常，可能会对视网膜产生严重后果。由MDR1基因编码的P-糖蛋白（P-gp）广泛表达于屏障组织和排泄组织， 表达于视网膜色素上皮（RPE）的神经视网膜和脉络膜侧，Aβ也被证实是P-gp底物之一。本项目旨在血视网膜屏障转运功能的角度，探讨早期AMD视网膜损伤的Aβ机制以及Aβ转运清除学说在AMD发病机制研究中的作用。.我们课题组近期的研究，验证了人RPE细胞具有活跃的P-gp转运活性，在高血糖和糖皮质激素等条件下可表现出不同的P-gp介导机制。我们应用低浓度Aβ刺激原代培养小鼠RPE细胞建立体外AMD模型，验证了低浓度Aβ可以抑制小鼠RPE细胞P-gp的表达以及转运活性。为进一步探讨P-gp在AMD氧化损伤状态下对血视网膜外屏障的作用，我们建立了血视网膜外屏障氧化应激模型来模拟AMD的病理状态，并发现通过氧化应激诱导的P-gp功能性表达增加减少血视网膜外屏障的线粒体损伤以及凋亡，提示P-gp在血视网膜外屏障的抗氧化损伤角色。.本项目的科学意义在于提出P-gp可能是干预早期年龄相关性黄斑变性中Aβ清除的潜在靶点。通过调节P-gp信号通路可以阐明血-视网膜屏障P-gp信号通路与早期AMD视网膜的Aβ增高和损伤的机制。这一新的研究切入点，不仅有望为早期AMD视网膜损伤和防治研究提供新的证据和研究思路，还将有助于阐明早期AMD视网膜的Aβ机制。