HIV-1 Tat蛋白对U87细胞活性及氧化应激的影响  

Effects of HIV-1 Tat protein on cell viability and oxidative stress of U87 cells

作　　者：郭帅志 李丹迪 秦泽明 温红玲 王志玉 黄涛[2] 赵丽 Guo Shuaizhi;Li Dandi;Qin Zeming;Wen Hongling;Wang Zhiyu;Huang Tao;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)

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

出　　处：《中华实验和临床病毒学杂志》2021年第1期34-38,共5页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的研究HAD和非HAD的艾滋病患者不同部位来源的Tat蛋白氨基酸位点变异及其对U87细胞氧化应激的影响。方法采用BLAST和MEGA6对一例HAD患者(H)和一例非HAD患者(N)的基底节(BG)、脾脏(SPL)共4个部位来源的HIV-1 Tat蛋白进行序列分析,研究其氨基酸位点变异,并将tat基因转染至U87细胞,显微镜观察绿色荧光蛋白(green fluorescent protein,GFP)初步判断Tat蛋白表达情况,Western blot检测细胞Tat蛋白的表达;并使用cck-8法、Western blot、MDA检测试剂盒研究不同部位Tat蛋白对细胞活性、氧化应激指标GPX、MDA水平的影响。结果氨基酸序列分析显示N-BG、N-SPL、H-BG、H-SPL来源的HIV-1 Tat蛋白关键氨基酸位点存在差异;Tat蛋白可抑制U87细胞的活性,抑制作用可以被抗氧化剂N-乙酰-L-半胱氨酸(NAC)逆转。与对照组相比,四个实验组MDA水平均升高,GPX蛋白表达量均降低,差异具有统计学意义(P=0.012,P=0.004,P=0.000,P=0.003;P=0.000,P=0.016,P=0.000,P=0.021);且不同部位的Tat蛋白诱导细胞氧化应激的能力不同,H-BG组MDA水平高于N-BG部位,差异具有统计学意义(P=0.023);且无论是HAD还是非HAD患者BG组GPX含量均低于SPL组,差异有统计学意义(P=0.01,P=0.007,P=0.01,P=0.007)。结论HAD及非HAD患者外周及中枢神经系统中的Tat蛋白关键氨基酸位点存在差异,对细胞氧化应激的影响也不同。Objective To study the amino acid site variation of HIV-1 Tat protein from different parts of AIDS patients with HAD and non HAD and its effect on oxidative stress of U87 cells.Methods HIV-1 Tat amino acid sequences were analyzed by BLAST and MEGA6 software to study the variation of amino acid sites in four parts of central nervous tissue basal ganglia(BG)and peripheral spleen(SPL)of an HIV-associated dementia(HAD)patient(H)and a non-HAD patient(N),The HIV-1 tat genes were transfected into U87 cell.The green fluorescent protein was observed under microscope to determine the Tat protein expression.The expression of Tat protein in U87 cells was detected by Western blotting.CK-8 method,Western blotting and malondialdehyde(MDA)detection kit were used to study the effect of Tat protein on cell activity,oxidative stress index glutathione peroxidase(GPX),MDA level.Results Amino acid sequence analysis showed that the key amino acid sites of HIV-1 Tat protein from N-BG,N-SPL,H-BG and H-SPL were different;Tat protein could inhibit the activity of U87 cells,which could be reversed by antioxidant N-acetyl-L-cysteine(NAC).Compared with the control group,the levels of MDA were increased and the expression of GPX protein was decreased in the four experimental groups(P<0.05).And different sources of Tat protein had different ability to induce oxidative stress,the level of MDA in H-BG group was higher than that in N-BG group(P<0.05).The expression of GPX protein in BG group of both HAD and non-HAD patients was lower than that of SPL group(P<0.05).Conclusions There are differences in the key amino acid sites of Tat protein in peripheral and central nervous system between HAD and non-HAD patients,and their effects on oxidative stress were also different.

关 键 词：HIV相关痴呆 HIV-1 Tat蛋白 丙二醛 谷胱甘肽过氧化物酶 

分 类 号：R512.91[医药卫生—内科学] R749.16[医药卫生—临床医学]