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【目的】抗反转录病毒疗法(ART)能够有效控制人免疫缺陷病毒(Human immunodeficiency virus-1,HIV-1)的复制,但是不能将其完全清除。至2012年底,全球仍有3 500万HIV-1感染者,同年约160万人死于艾滋病(Acquired immune deficiency syndrome,AIDS)及其相关疾病。HIV-1感染难以根治的主要原因之一是机体内HIV-1潜伏储存库(Reservoir)的存在。HIV-1潜伏储存库主要由CD4+T细胞和单核巨噬细胞构成,与CD4+T细胞相比,目前研究者对单核巨噬系细胞中HIV-1病毒复制机制尚不明了,且缺乏适宜的研究体系。因此,为探讨单核细胞活化或分化信号对HIV-1复制的影响,我们建立了旨在研究HIV-1前病毒转录调控机制的人单核巨噬细胞系模型。【方法】构建env区域移码突变和nef区域携带EGFP或Nano Luc报告基因的HIV-1 NLn GFP-Kp或NLn Nano Luc-Kp重组病毒,分别感染2种人单核细胞系THP-1或U937细胞。通过有限稀释法制备单克隆细胞系,利用流式细胞术或Nano Luc荧光素酶活性分析检测报告基因的表达。筛选EGFP或Nano Luc阴性表达的细胞克隆,经激活剂佛波酯(Phorbol-12-myristate-13-acetate,PMA)刺激后鉴定潜伏感染的细胞克隆。【结果】研究中鉴定了4个HIV-1潜伏感染的细胞克隆。其中2个是表达EGFP的THP-1克隆,2个是以Nano Luc为报告基因的U937克隆。这些克隆在PMA刺激处理后皆有报告基因的表达,而在恒态条件下未检测到报告基因的表达。【结论】成功建立了4个HIV-1潜伏感染的人单核细胞系克隆,该模型有助于理解单核巨噬系细胞的HIV-1病毒复制机制,可能成为进一步研究HIV-1前病毒转录调控机制的有力工具。
【Aim】 Anti-retroviral therapy (ART) can effectively control the replication of human immunodeficiency virus-1 (HIV-1), but it can not be completely eliminated. By the end of 2012, there were still 35 million HIV-1 infected people in the world. About 1.6 million people died of Acquired Immune Deficiency Syndrome (AIDS) and related diseases in the same year. One of the main reasons why HIV-1 infection is difficult to cure is the presence of HIV-1 latent reservoirs in the body. The HIV-1 latent repositories are mainly composed of CD4 + T cells and mononuclear macrophages. Compared with CD4 + T cells, the mechanism of replication of HIV-1 virus in mononuclear macrophage-derived cells remains unclear. Lack of suitable research system. Therefore, in order to investigate the effect of monocyte activation or differentiation signaling on HIV-1 replication, we established a human monocyte-macrophage cell line model to study the transcriptional regulation of HIV-1 provirus. 【Method】 HIV-1 NLn GFP-Kp or NLn Nano Luc-Kp recombinant viruses carrying frame coding mutations in the env region and nef region carrying EGFP or Nano Luc reporter genes were constructed and infected with two human monocytic cell lines, THP-1 and U937 cell. Monoclonal cell lines were prepared by limiting dilution and reporter gene expression was detected by flow cytometry or Nano Luc luciferase activity assay. The cell clones negative for EGFP or Nano Luc were screened, and the latent infected cell clones were identified after stimulation with activator phorbol-12-myristate-13-acetate (PMA). [Results] Four HIV-1 latent infected cell clones were identified in the study. Two of them were THP-1 clones expressing EGFP and two were U937 clones carrying Nano Luc as the reporter gene. These clones had reporter gene expression after PMA stimulation, whereas no reporter gene expression was detected under the constant state. 【Conclusion】 Four HIV-1 latent human monocytic cell clones were successfully established. This model is helpful to understand the mechanism of HIV-1 virus replication in mononuclear macrophage-derived cells and may be useful for further research on HIV-1 provirus A powerful tool for transcriptional regulation.