ApoE促进LRP1介导小胶质细胞内化降解Aβ的作用研究

项目名称： ApoE促进LRP1介导小胶质细胞内化降解Aβ的作用研究

项目编号： No.81500928

项目类型： 青年科学基金项目

立项/批准年度： 2016

项目学科： 医药、卫生

项目作者： 杨维娜

作者单位： 西安交通大学

项目金额： 17.5万元

中文摘要： 促进小胶质细胞内化降解Aβ是减少AD患者脑内Aβ沉积的有效途径。本研究拟首先培养apoe-/-鼠小胶质细胞和星形胶质细胞，利用定点突变及基因克隆技术构建携带人源性apoeε2、ε3、ε4基因片段的慢病毒颗粒并转染星形胶质细胞，收集条件培养基培养小胶质细胞，免疫荧光染色及ELISA方法筛选出与细胞内化降解Aβ最为相关的ApoE亚型；其次，构建LRP1过表达和RNA干扰慢病毒颗粒，在细胞和整体水平上调和下调LRP1后观察细胞内化Aβ的变化，Morris水迷宫检测AD转基因鼠学习记忆力的改变；再次，在细胞水平应用各种蛋白酶抑制剂研究内化入溶酶体内Aβ的降解途径；最后，应用PI3K抑制剂阻断PI3K/Akt通路，在细胞和整体水平观察Aβ的变化。通过该研究阐明ApoE对LRP1介导小胶质细胞内化降解Aβ的促进作用及PI3K/Akt通路对该过程的调控作用，并为临床治疗AD提供新的实验依据和理论基础。

中文关键词： 阿尔茨海默病；β-淀粉样蛋白；载脂蛋白E；低密度脂蛋白受体相关蛋白1；PI3K/Akt

英文摘要： Alzheimer’s disease (AD) is characterized by the accumulation and deposition of β-amyloid (Aβ) peptides within the brain, leading to the perturbation of synaptic function and neuronal loss that typifies the disease. Microglial internalization and degradation of Aβ are believed to be key mechanisms of the initial defense of the brain against the toxic accumulation of Aβ. Therefore, promoting microglia activation may be a powerful strategy to clear Aβ and prevent Aβ-associated pathological events and neuronal loss. In this study, we use immunoelectron microscopy, confocal microscopy, ELISA, immunoprecipitation, Western blotting, gene cloning, RNAi, and Morris water maze to investigate astroglial apolipoprotein E promotes low density lipoprotein receptor-related protein 1 (LRP1) mediated beta-amyloid protein internalization and degradation in microglia, and to assess phosphatidylinositol 3 kinase/Akt(PI3K/Akt) signaling pathways involving the regulation of Aβ internalization and degradation. First, astrocytes and microglia were obtained from postnatal apoe knock-out mouse (apoe-/-) by a standard enzyme treatment protocol. The astrocyte and microglia purity were determined by glial fibrillary acidic protein (GFAP) and ionizing calcium adapter binding molecule 1(Iba1) positivity respectively through immunofluorescence. Recombinant plasmid that carrying each apoe genotype (ε2, ε3, ε4) were transfected into apoe knocked out astrocytes. Then microglia was exposed to conditioned media from astrocytes. Choose an ApoE subtype that significant promotes Aβ internalization and degradation in microglia. Second, to confirm the effect of LRP1 on Aβ internalization and degradation in microglia, we observe Aβ level after overexpressing LRP1 and knockdown LRP1, and to evaluate this effect on learning and memory in AD transgenic mice. Third, inhibitors of neprilysin (NEP) , insulin degradation enzymes (IDE), lysosomal proteases and proteasome were used to study the intracellular Aβ degradation machinery. Fourth, we observe Aβ level after inhibitions of PI3K/Akt signaling pathway. Our study will provide valuable data and theory foundation for the pathogenesis and treatment of AD.

英文关键词： Alzheimer’s disease;beta-amyloid protein;apolipoprotein E;low-density lipoprotein receptor-related protein 1;PI3K/Akt