视网膜血管瘤样增生（RAP）动物模型建立和发病机制研究

Age-related macular degeneration (AMD) is the most common cause of legal blindness in the elders worldwide. Neovascular AMD accounts for 90% of legal blindness attributable to AMD, which can be classified into three subtypes, namely typical choroidal neovascularization (CNV), polypoidal choroidal vasculopathy (PCV), and retinal angiomatous proliferation (RAP). The most common clinical features of RAP include rapid loss of vision，retinal and preretinal hemorrhages as well as pigment epithelial detachments, and can be divided into 3 clinical stages. RAP resists various treatments and recurs persistently. The mechanism of RAP is largely unknown. Pathologically RAP was a circumscribed intraretinal angiomatous complex, like retinal hemangioma, including stromal cells and endothelium cells. RAP stromal cells express high levels of VEGF. Retinal hemangioma is caused by VHL gene mutation. VHL protein is one part of an E3 ubiquitin ligase complex that ubiquitylates HIF-a subunit leading to rapid degradation. HIF is a transcription factor can activate various angiogenesis factors such as VEGF. In our previous work, we had established an inheritable RAP mouse model by knocking out vhl gene and cell cycle regulators Rb/p107 genes in the retina using pax6 alpha-enhancer Cre (a-Cre). This model can mimic all 3 stages of RAP, and also has retinal pigment epithelial cell degeneration and detachment. Preliminary data indicated that the RAP may be related to retinal cell fate switch, E2f1-depedent stromal cell proliferation, Hif-induced pro-angiogenic factors production, oxidative stress induced RPE cell death. This model provides an excellent opportunity for us to study RAP in vivo. We will focus on the following 3 aims. Aim 1 to characterize the pathogenesis, histopathological and imaging features of this model, including FFA (fundus flourescein angiography) and ICGA (indocyanine green angiography). Aim 2 to explore the mechanism of RAP, including the cell origin of the RAP stromal cells, RNA microarray analysis of RAP to determine the major gene expression pattern changes. Aim 3 we will test two Hif1a inhibitors including Topotecan and Digoxin to prevent or treat RAPs.

视网膜血管瘤样增生（RAP）是新生血管性老年黄斑变性的一个重要亚型，发病机制不清楚，治疗困难，缺乏合适的动物模型。在前期的研究中，我们通过在小鼠视网膜同时敲除Vhl基因和Rb/p107基因，初步建立了一个可模拟人RAP主要病理特征的动物模型；提示缺氧调节和细胞周期通路同时失调是RAP发生的重要机制，可能与视网膜细胞命运转换、基质细胞持续分裂、促血管因子分泌、RPE细胞程序性坏死等有关。拟对这一独特动物模型的临床特征进行观察鉴定，并探讨RAP的发生机制和防治。将从3个方面进行：1-动物模型鉴定，包括发病过程、组织病理、电镜及影像学特征分析；2-发病机制研究，包括基质细胞起源、基因表达谱分析；3-药物试验，测试缺氧诱导因子抑制剂拓扑替康和地高辛对RAP的防治效果。建立可靠的RAP动物模型，有助于我们全面理解老年黄斑变性的病理过程，对于揭示其发生发展的分子机制、设计合理的治疗方案具有重要的价值。

视网膜血管瘤样增生（RAP）是新生血管性老年黄斑变性（AMD）的一个重要亚型。本项目主要研究一个我们自主建立的小鼠RAP模型。Rb基因缺失可以导致儿童视网膜母细胞瘤，Vhl基因突变可导致视网膜毛细血管瘤；我们发现Rb蛋白家族和Vhl信号通路在RAP发病过程中均有重要作用。本研究的主要研究内容和结果如下： (1) 发现Rb/Vhl双敲除视网膜可形成大量新生血管，而单纯敲除Rb基因或Vhl基因都导致视网膜血管发育障碍。在Vhl-/-的基础上敲除一个拷贝的Rb基因也可导致视网膜新生血管，表明Rb蛋白对Vhl-/-血管表型的抑制有剂量效应。 (2) 发现Rb/Vhl双敲除视网膜可导致光感受器自噬增强而死亡，Rb基因敲除和Vhl基因敲除对视杆视锥细胞有协同致死作用。Vhl基因敲除可导致视网膜自噬轻度增强，Rb基因敲除可导致部分视杆细胞凋亡。Rb蛋白可抑制Vhl-/-导致的自噬，并有剂量效应。(3) 发现Rb蛋白可结合于多个Hif靶基因的启动子（Epo, Vegfa, Vegfr2, Tie2, Bnip3），荧光素报告试验证实Rb蛋白可不同程度地抑制这些基因的转录活性。RT-PCR也发现Rb蛋白可不同程度地抑制这些基因的表达水平。(4) 通过敲除CyclinD1激活Rb蛋白，可以导致视网膜表层血管发育延缓，证明Rb蛋白对Vhl-/-的血管表型的抑制是必需而且充分的。（5）Rb/p107双敲除视网膜可以形成视网膜母细胞瘤，虽然Vhl基因也是一个经典的肿瘤抑制基因，但Vhl-/-抑制了Rb/p107双敲除导致的、来源于无长突细胞的视网膜母细胞瘤，但却促进了形成基质细胞来源于Müller胶质细胞的RAP；RAP位于视网膜下腔，结构上和RCH类似。（6）RNA测序发现RAP组织下调了细胞周期、DNA修复信号通路，但上调了Hif、PI3K、补体、细胞外间质通路。.本研究阐明了Rb蛋白的一个新功能（抑制Hif靶基因的表达），建立了一个可模拟人类AMD多个危险因素的RAP模型，如吸烟（可导致Rb蛋白失活）、氧化应激（可激活Hif通路）、涉及补体系统、细胞外间质的稳态维持的遗传因素（主要与染色体1q32和10q26上的基因变异有关）。首次明确了RAP的基质细胞来源于Müller胶质细胞。