HIV-1潜伏感染人Jurkat T细胞模型的建立  

Establishment of a HIV-1 latent infection model in Jurkat T cell line

作　　者：李琳[1] 仇超[2,3] 李亮助[2,3] 刘爱平[2,3] 邱趁丽[2,3] 周明哲[2,3] 张晓燕[2,3,4,5] 徐建青[2,3,4,5] 朱焕章[1] 

机构地区：[1]复旦大学生命科学学院遗传学研究所遗传工程国家重点实验室,上海200433 [2]复旦大学附属上海市公共卫生临床中心 [3]复旦大学生物医学研究院,上海201508 [4]复旦大学上海医学院分子病毒学重点实验室,上海200032 [5]中国疾病预防控制中心传染病预防控制国家重点实验室,北京102206

出　　处：《中国艾滋病性病》2011年第4期387-390,394,共5页Chinese Journal of Aids & STD

基　　金：国家十一五重大专项《艾滋病和病毒性肝炎等重大传染病防治研究项目》资助(课题编号2008ZX10001-006)~~

摘　　要：目的建立Ⅰ型艾滋病病毒(HIV-1)潜伏感染人Jurkat T细胞的模型,为进一步研究HIV-1潜伏感染机制奠定基础。方法利用一种表达增强型绿色荧光蛋白(EGFP)的HIV-1假病毒感染人Jurkat T细胞,采用流式细胞术分选出GFP-细胞群,之后经激活剂曲古抑菌素A(Trichostatin A,TSA)刺激后,再次采用流式细胞术分选出GFP+细胞,并通过有限稀释法制备获得单克隆细胞系。利用流式细胞术及酶联免疫吸附试验(ELISA)分别检测单克隆细胞系在TSA刺激前后的GFP+细胞比例及HIV-1病毒抗原p24;并用Alu-长末端重复序列(LTR)套式聚合酶链反应(Nested PCR)检测单克隆细胞系中HIV-1基因组的整合情况。结果获得了5株HIV-1潜伏感染的Jurkat单克隆细胞系,对这5株单克隆细胞系分别进行激活剂处理,发现无论是GFP表达细胞比例或HIV-1病毒蛋白p24抗原的表达,皆显著高于激活剂处理前的背景表达水平(P<0.05);并能从这5个单克隆细胞系基因组中扩增出针对HIV-1基因组的特异条带。结论已获得的5株单克隆细胞系具有HIV-1潜伏细胞的生物学特点,表明已成功地建立了HIV-1潜伏感染细胞模型。该模型可用于各种药物或激活剂的筛选和检测,也为进一步研究HIV-1潜伏感染机制奠定了工作基础。Objective To establish a HIV-1 latent infection model in human Jurkat T cell line,which will be used for the mechanism study of HIV-1 latency.Method HIV-1 pseudoviruses which express Enhanced Green Fluorescent Protein（EGFP） were prepared to infect human Jurkat T cells.After sorting out GFP-cell population by Fluorescence-Activated Cell Sorting（FACS）,potential latent clones were screened by the detection of reactivation of GFP after Trichostatin A（TSA） stimulation.These GFP reactivated cell populations were isolated by FACS and cloned to cell lines by limiting dilution.Further test was performed for GFP＋ cell percentage by Flow Cytometry and HIV-1 p24 expression by ELISA before and after stimuli for the clone lines,as well as Alu-LTR nested PCR for HIV-1 genome integration test.Result Five HIV-1 latently infected Jurkat T cell lines were gained,of which GFP and p24 expression were extremely low under basal conditions while significantly raised after TSA stimulation.Alu-LTR nested PCR assay also gained specific band of HIV-1 genomes.Conclusion The 5 clone lines had the biological characteristics of HIV-1 latently infected cells,indicating that HIV-1 latent infection in vitro model was successfully established,which could be used for the screening and detection of drugs or stimuli,as well as for better serving the mechanism study of HIV-1 latency.

关 键 词：艾滋病病毒Ⅰ型 JURKAT T细胞系 绿色荧光蛋白 潜伏感染模型 

分 类 号：R512.91[医药卫生—内科学]