HAD及非HAD患者中枢神经系统来源的HIV-1 Nef蛋白对U87细胞自噬的影响  

Effect of HIV-1 Nef protein on autophagy of U87 cells in HAD and non-HAD patients

作　　者：单晓宇 曹馨月 郑文慧 郭帅志 温红玲[1] 王志玉[1] 黄涛[2] 赵丽[1] Shan Xiaoyu;Cao Xinyue;Zheng Wenhui;Guo Shuaizhi;Wen Hongling;Wang Zhiyu;Huang Tao;Zhao Li(Department of Laboratory Science of Sanitary Microbiology,School of Public Health,Shandong University,Key Laboratory of Shandong Province During the 13th Five-year Plan,Jinan 250012,China;Shandong Provincial Center for Disease Control and Prevention,Jinan 250014,China)

机构地区：[1]山东大学公共卫生学院卫生微生物检验学系,山东省"十三五"高校重点实验室,济南250012 [2]山东省疾病预防控制中心,济南250014

出　　处：《中华实验和临床病毒学杂志》2020年第1期7-11,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的研究HIV-1 Nef蛋白氨基酸位点变异对其抑制神经细胞自噬的影响,探讨Nef蛋白致中枢神经系统损伤的机制。方法分别扩增和克隆1例HIV相关性痴呆(HIV-associated dementia,HAD)患者(H)及1例非AIDS HAD患者(N)中枢神经系统颞叶(temporal cortex,TC)部位的HIV-1 nef基因,进行序列分析,研究氨基酸位点变异;构建H-TC和N-TC来源的Nef真核表达载体p EGFP-N1-nef,并分别转染U87细胞,观察绿色荧光蛋白并初步判断Nef蛋白表达情况;同时Western blot检测U87细胞Nef蛋白及细胞自噬标记蛋白LC3-Ⅱ、p62蛋白的表达,分析不同来源的Nef对U87细胞自噬的影响。结果PCR扩增并构建H-TC及N-TC nef基因克隆载体pMD-19T-nef,测序证实为HIV-1B亚型,氨基酸序列分析显示,H-TC与N-TC关键氨基酸位点存在差异;成功构建pEGFP-N1-nef重组质粒,在U87细胞内表达Nef蛋白,转染后48 h Nef蛋白表达量最高;Western blot结果分析表明,LC3-Ⅱ蛋白的表达量在组间的差异有统计学意义(F=11.764,P=0.001),两组细胞中LC3-Ⅱ含量较低,Western blot未能检测到。,空白组与空载体组之间LC3-Ⅱ的表达量差异无统计学意义(P=0.169),H-TC、N-TC及阳性对照CQ组LC3-Ⅱ表达升高,与空白对照或空载体相比,差异具有统计学意义(P=0.017,P=0.039,P=0.031),且H-TC组LC3-Ⅱ的表达量高于N-TC组(P=0.023);H-TC、N-TC及阳性对照CQ组p62蛋白的表达量较空白对照或空载体组有所升高,但组间差异无统计学意义(F=2.049,P=0.163)。结论HAD及非HAD患者中枢神经系统中HIV-1 Nef氨基酸位点存在差异,对U87细胞自噬的影响也因此不同,H-TC来源的Nef诱导自噬标记蛋白LC3-Ⅱ表达的能力更强。Objective To study the effect of amino acid site variation of HIV-1 Nef protein on its inhibition of neuronal autophagy and explore the mechanism of Nef protein-induced central nervous system injury.Methods HIV-1 nef genes were amplified and cloned from the temporal cortex(TC)of the central nervous system in 1 case of HIV-associated dementia(HAD)H and 1 case of non-HAD AIDS patient N.The amino acid sequences were aligned by NCBI BlAST tools and MEGA6.0 software to study the variation of amino acid sites.The eukaryotic expression vectors pEGFP-N1-nef derived from H-TC and N-TC were constructed and transfected into U87 cells to observe green fluorescence.At the same time,the expression of Nef protein,LC3-Ⅱand p62 protein in U87 cells were detected by Western blot,and the effects of different sources of Nef on autophagy of U87 cells were analyzed.Results The nef genes were amplified by PCR and clone vectors pMD-19T-nef of H-TC and N-TC were successfully constructed.The sequencing confirmed that they were HIV-1B subtypes.The amino acid sequence analysis showed that there were differences between H-TC and N-TC key amino acid sites.The recombinant plasmid pEGFP-N1-nef was successfully constructed and expressed Nef protein in U87 cells.Western blot analysis showed that the expression of LC3-II protein was significantly different among groups(F=11.764,P=0.001).There was no significant difference in the expression of LC3-II between cell control and plasmid control(P=0.169).The content of LC3-Ⅱwas low in the two groups of cells,which could not be detected by Western blot.The expression of LC3-II in H-TC,N-TC and positive control CQ group increased,compared with blank control or blank vector.The difference was statistically significant(P=0.017,P=0.039,P=0.031),and the expression of LC3-II in H-TC group was higher than that in N-TC group(P=0.023);the expression of p62 protein in H-TC,N-TC and positive control CQ group was higher than that in blank control or blank vector group,but there was no significant difference betw

关 键 词：HIV相关性痴呆 HIV-1 NEF 自噬标记蛋白LC3-Ⅱ 自噬标记蛋白p62 

分 类 号：R74[医药卫生—神经病学与精神病学]