HAD患者来源的HIV-1 Nef蛋白对U87细胞分泌TNF-α和IL-1β细胞因子的影响  

Effect of HIV-1 Nef proteins from a patient with HIV-1-associated dementia on the secretion of TNF-αand IL-1βcytokines by U87 cells

作　　者：李丹迪 秦泽明 贺淑婷 温红玲 黄涛[2] 张克胜 赵丽 Li Dandi;Qin Zeming;He Shuting;Wen Hongling;Huang Tao;Zhang Kesheng;Zhao Li(Department of Laboratory Science of Microbiology,School of Public Health,Shandong University,Key Laboratory of Shandong Province During the 13th Five-year Plan,Jinan 250012,China;Shandong Center for Disease Control and Prevention,Jinan 250014,China;Comprehensive Supervision and Law Enforcement Bureau of Shibei District Health Bureau,Qingdao 266000,China)

机构地区：[1]山东大学公共卫生学院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 [3]青岛市北区卫健局综合监督执法局,266000

出　　处：《中华实验和临床病毒学杂志》2021年第3期241-245,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的探讨HIV-1负调控因子(Nef)在HIV-1神经致病性中的作用。方法从HIV-1相关痴呆(HIV-associated dementia,HAD)患者的中枢神经系统(central nervous system,CNS)和外周5个部位(基底结、额叶皮质、脑膜、颞叶皮质和脾)中获得5株不同的HIV-1 nef基因,与载体pcDNA3.1连接构建重组的pcDNA3.1-nef真核表达载体,转染人神经胶质瘤细胞U87。转染后22 h、27 h、32 h、37 h和42 h经免疫组化法和Western blot检测Nef蛋白表达情况,采用JEDA801D和JD-801软件对结果进行半定量分析。转染后27 h~62 h,每间隔5 h收集U87细胞的培养上清,ELISA测定上清中两种细胞因子TNF-α和IL-1β的含量,分析两种细胞因子的动态表达情况。结果成功构建5株来自一例HAD患者不同部位组织的nef基因的重组的pcDNA3.1-nef真核表达载体,转染U87细胞。免疫组化实验的结果显示,Nef蛋白在转染后42 h开始表达,Western blot进一步证实了这一结果。ELISA结果显示,转染后22 h、27 h、32 h、37 h,各组上清中细胞因子的含量随时间逐渐增多,但各组间的差异均无统计学意义(TNF-α:F=0.445,P=0.837;F=0.579,P=0.742;F=0.617,P=0.714;F=2.728,P=0.057。IL-1β:F=2.656,P=0.062;F=0.485,P=0.809;F=0.165,P=0.982;F=2.463,P=0.093);42 h后,各组细胞因子含量逐渐降低,57 h~62 h,TNF-α及IL-1β含量基本维持稳定;转染后42 h、47 h、52 h、57 h、62 h,实验组两种细胞因子的含量明显高于对照组,差异有统计学意义(TNF-α:F=241.310,P<0.001;F=242.638,P<0.001;F=250.114,P<0.001;F=143.877,P<0.001;F=146.172,P<0.001。IL-1β:F=251.578,P<0.001;F=188.816,P<0.001;F=276.240,P<0.001;F=238.136,P<0.001;F=163.361,P<0.001),各对照组间或实验组间的差异无统计学意义(P>0.05)。结论HIV-1 Nef蛋白能诱导并增强U87细胞分泌TNF-α和IL-1β的能力,而HAD患者体内不同来源的HIV-1 Nef蛋白发生氨基酸变异对U87细胞分泌TNF-α和IL-1β无影响。Objective To investigate the role of HIV-1 negative regulator(Nef)in HIV-1 neuropathogenicity.Methods Five different HIV-1 nef genes were obtained from the central nervous system(CNS)and peripheral regions(basal ganglia,frontal cortex,meninges,temporal cortex and spleen)of a patient with HIV-1-associated dementia(HAD).The recombinant pcDNA3.1-nef eukaryotic expression vectors were constructed by connecting them with pcDNA3.1 vector and transfected into human glioma cell line U87 respectively.The expression of Nef protein was detected by immunohistochemistry and Western blotting at 22ndhour,27 th hour,32nd hour,37th and 42nd hour after transfection.The result were analyzed quantitatively by JEDA801D and JD-801 software.The supernatant of U87 cells was collected every 5 hours from 27th hour to 62nd hour after transfection.The levels of TNF-αand IL-1βin the supernatant were determined by ELISA,and the dynamic expression of the two cytokines was analyzed.Results Five recombinant pcDNA3.1-nef eukaryotic expression vectors of nef genes from different tissues of an HAD patient were successfully constructed and transfected into U87 cells.The result of immunohistochemistry showed that Nef protein began to express at 42nd hour after transfection,which was further confirmed by Western blot.The result of ELISA showed that the levels of cytokines in the supernatant of each group increased gradually with time from 22ed hour to 37th hour after transfection,but there was no significant difference among the groups(TNF-α:F=0.445,P=0.837;F=0.579,P=0.742;F=0.617,P=0.714;F=2.728,P=0.057.IL-1β:F=2.656,P=0.062;F=0.485,P=0.809;F=0.165,P=0.982;F=2.463,P=0.093);The levels of TNF-αand IL-1βin the experimental group were significantly higher than those in the control group from the 42nd hour(P<0.05);after 42nd hour,the levels of cytokines in each group gradually decreased,and the levels of TNF-αand IL-1βremained stable from the 57th hour to the 62nd hour,while the levels of TNF-αand IL-1βin the experimental group were higher than

关 键 词：HIV-1 HIV-1相关痴呆 HIV-1 Nef蛋白 TNF-Α IL-1Β 

分 类 号：R747.9[医药卫生—神经病学与精神病学] R512.91[医药卫生—临床医学]