N2a/APP695细胞表达载脂蛋白E4对线粒体自噬的影响  

Effects of apolipoprotein E4 expression on mitophagy in N2a/APP695 cells

作　　者：付燕林 吴昌学[1] 郑菊 龙培艳 张文萍 高霄 王正微 齐晓岚[1] 肖雁[1] FU Yanlin;WU Changxue;ZHENG Ju;LONG Peiyan;ZHANG Wenping;GAO Xiao;WANG Zhengwei;QI Xiaolan;XIAO Yan(Key Laboratory of Endemic and Minority Diseases of Ministry of Education&Guizhou Provincial Key Laboratory of Medical Molecular Biology,Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学地方病与少数民族疾病教育部重点实验室&贵州省医学分子生物学重点实验室,贵州贵阳550004

出　　处：《贵州医科大学学报》2022年第5期497-503,共7页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81660207);贵州省科技厅基础重点项目(黔科合基础〔2019〕1440)。

摘　　要：目的探讨载脂蛋白E4(APOE4)表达对N2a/APP695细胞中线粒体自噬的影响。方法慢病毒APOE4感染N2a/APP695细胞构建表达APOE4细胞组(表达组),同时设立N2a/APP695正常细胞组(正常组)、阴性对照慢病毒空载N2a/APP695细胞组(阴性对照组),共3个实验组;培养72 h后进行嘌呤霉素筛选处理,Western blot检测细胞APOE4表达情况、线粒体融合蛋白-2(MFN2)、MFN1及线粒体分裂蛋白-1(FIS1)、线粒体自噬相关蛋白PINK1、Parkin及微管相关蛋白1轻链3(LC3)的表达,激光共聚焦观察线粒体形态及细胞中胆固醇含量,免疫荧光观察线粒体与LC3Ⅱ的共定位情况。结果成功构建表达APOE4的慢病毒感染N2a/APP695细胞;与正常组和阴性对照组相比,表达组MFN2、MFN1蛋白表达差异无统计学意义(P>0.05),FIS1、PINK1、Parkin蛋白表达明显上调(P<0.01),LC3Ⅱ/Ⅰ比值下调(P<0.05),线粒体形态碎片化和细胞中胆固醇高,LC3Ⅱ与线粒体的共定位减少。结论表达APOE4的N2a/APP695细胞中线粒体分裂增多、线粒体自噬关键通路障碍、自噬异常,这可能与APOE4减少胆固醇外排、细胞内游离胆固醇增多、进而破坏线粒体分裂融合平衡有关。Objective To investigate the effect of apolipoprotein(APOE4)expression on mitophagy in N2a/APP695 cells.Methods Lentivirus APOE4 infected N2a/APP695 cell line to construct APOE4 cell group(expression group),and the N2a/APP695 cells were divided into normal cell group(normal group),negative control lentivirus empty vector N2a/APP695 cell group(negative control group),which were screened by puromycin and then cultured for 72 h;Western blot was used to detect the expression of APOE4,mitochondrial fusion proteins MFN2,MFN1 and fission protein FIS1,mitophagy-related proteins PINK1,Parkin,and LC3;laser confocal was adopted to observe mitochondrial morphology and cholesterol content in cells;immunofluorescence was adopted to observe mitochondria colocalization with LC3Ⅱ.Results APOE4 expression lentivirus infected N2a/APP695 cell was successfully constructed.Compared with normal group and negative control group,MFN2 and MFN1 protein expression differences were not statistically significant(P<0.05);FIS1,and PINK1,and Parking protein expression were obviously upregulated(P<0.01);LC3Ⅱ/Ⅰratio was down-regulated(P<0.05);mitochondrial was morphologically fragmented and cholestral was high within cell,LC3Ⅱshared less co-localization with mitochondrial.Conclusion APOE4 expressed N2a/APP695 mitochondrial shows more mitochondrial fission,key pathway barrier in mitophagy,abnormal mitophagy;this may affect the balance of mitochondrial fission and fusion by increasing free cholesterol and APOE4 reduce cholestral discharge.

关 键 词：阿尔茨海默病 载脂蛋白E 线粒体自噬 胆固醇 

分 类 号：R34-33[医药卫生—基础医学]