胆固醇代谢通路在黄斑基底膜线性沉积及软性玻璃膜疣形成中的作用及调控靶点研究

Basal linear deposit (BLinD) and soft drusen are deposits of lipoproteins on Bruch membrane, containing esterified cholesterol. They are the pathologic changes specific for age-related macular degeneration (AMD), also are the basis of AMD progression. The major component of BLinD and soft drusen are cholesterol origined from diet. The polymorphisms of cholesterol-related genes are significantly associated with AMD, but not affecting the cholesterol level in blood, which suggests local dysregulation of cholesterol metabolism in retina is key to the development of AMD. But the localizations and functional changes of cholesterol-related protein in AMD are not yet clear. Herein, we aim to detect these changes by comparing the cholesterol-related proteins and lipids in the retina from AMD patients and controls. With these information, we will further construct vectors by CRISPR-Cas9, and establish an APOE*3-Leiden.CETP mouse model with local cholesterol metabolism defect. The pathogenesis of this model will be illustrated by measuring related changes in retina, including lipid, lipoprotein, apolipoprotein, uptakes of HDL and LDL, and expression of genes in the downstream and upstream of the targeted gene. Potential target for gene therapy of BLinD and soft drusen will therefore be decided. Meanwhile, subretinal injection of targeted gene and intravitreal injection of apoA-I mimic peptide will be applied to the Macaca fascicularises model induced with cholesterol-rich diet. Their effect on clearing BLinD and soft drusen, and the associated mechanism will be studied. We expect that the mechanism of BLinD and soft drusen formation and effective treatment methods will be learned in the end.

基底膜线性沉积(BLinD)和软性玻璃膜疣是老年黄斑变性(AMD)特有的病理改变和进展基础；其本质是富含酯化胆固醇的脂蛋白混合物,主要来自食源性胆固醇。胆固醇相关基因多态性与AMD显著相关，但不影响患者血清胆固醇水平，提示局部视网膜胆固醇调控缺陷才是AMD发病关键，其缺陷的细胞定位和机制不清。本研究拟对人干性AMD视网膜脉络膜组织进行胆固醇代谢相关基因表达谱测序和脂质谱分析，免疫荧光定位差异蛋白所在细胞及细胞器，结合生物信息学分析，得出关键差异基因位点；进而CRISPR-cas9编辑对应基因位点，利用人源CETP转基因小鼠建立局部视网膜胆固醇代谢缺陷模型，研究其致病机制和开发基因靶点药物；同时在本课题组合作构建的高脂饮食诱导食蟹猴干性AMD模型玻璃体腔注射有效的基因靶点药物和ApoA-I类似物，验证其对BLinD和软性玻璃膜疣的清除作用，以期阐明AMD的始动病理改变成因和提供治疗靶点。

基底膜线性沉积和软性玻璃膜疣是AMD的重要致病机制。本项目通过食蟹猴模型观察到其年龄相关性眼底改变，及其与人类年龄相关性眼底改变的异同，为采用食蟹猴为动物模型的AMD研究提供了重要依据。其中，食蟹猴不同于人类的眼底改变为：更常见的黄色点状黄斑病变和眼底强自发荧光病灶，视网膜下玻璃膜疣样沉积、基底膜沉积、地图样萎缩和脉络膜新生血管表型的缺失；相同点在于：均有硬性玻璃膜疣、软性玻璃膜疣、视网膜动脉硬化、豹纹状眼底、周边脉络膜血管ICGA晚期染色和周边玻璃膜疣。通过进一步组织病理研究提示，眼底强自发荧光病灶对应于RPE下玻璃膜疣样沉积。具有黄色点状黄斑病变和硬性玻璃膜疣的食蟹猴具有更高的BMI；然而，高脂高糖饮食组与正常饮食组食蟹猴在以上表型的患病率及严重程度上均无显著差异，高脂高糖饮食对食蟹猴眼底表型的影响可能需要更长时间的随访观察（>2年）。此外，本项目研究发现，基底膜线性沉积是PCV和特发性浆液性PED的重要发病机制，提示以清除基底膜线性沉积或软性玻璃膜疣为靶点的治疗，具有广泛的应用价值。