【作者】 曾怡；

【导师】 卢春；

【作者基本信息】 南京医科大学 ， 微生物学， 2004， 硕士

【摘要】 本文旨在研究人类免疫缺陷病毒Ⅰ型(human immunodificency virus 1，HIV-1)Tat蛋白协同人类疱疹病毒6型(human herpesvirus 6,HHV-6)对人类疱疹病毒8型(human herpesvirus 8,HHV-8)裂解周期复制的影响。本研究将HHV-6 U1102株感染的JJhan细胞与HHV-8潜伏感染的BCBL-1细胞进行细胞融合，采用RT-PCR和RealTime RT-PCR对HHV-8裂解周期基因ORF26的转录水平进行检测。结果显示，细胞融合后实验组ORF26转录水平高于对照组(t检验，P＜0.05)。进而将HHV-6 U1102株感染的JJhan细胞与HHV-8潜伏感染的BCBL-1细胞进行混合共培养，并以HHV-6感染细胞培养上清作为条件培养基培养BCBL-1细胞，RealTime RT-PCR检测ORF26的转录水平和ELISA检测培养上清中IFN-γ和IL-10水平。结果显示，混合共培养中实验组ORF26的转录水平高于对照组(t检验，P＜0.05)。灭活前后的HHV-6感染细胞培养上清培养BCBL-1细胞均能上调ORF26转录水平(t检验，P＜0.05)；混合共培养和条件培养基培养的实验组与对照组细胞培养上清中IL-10的表达水平没有差异(t检验，P＞0.05)，混合共培养的实验组细胞培养上清中IFN-γ水平高于对照组(t检验，P＜0.05)，但条件培养基培养的实验组与对照组细胞培养上清中IFN-γ水平没有差异(t检验，P＞0.05)。56℃和紫外线灭活前后的纯化的HHV-6病毒颗粒感染BCBL-1后，分别在不同时间点开始出现高于对照组的ORF26转录。应用基因转染和Transwell共培养系统分别研究胞内和胞外表达的HIV-1 Tat蛋 南京医科人学硕_}学位论文白与HHV-6组合对ORF26转录水平的影响;评价胞内Tat对HHV-8复制的影响C结果显示，单纯细胞内外表达的Tat蛋白不能上调ORF26转录水平，但是胞内表达的Tat蛋白协同HHV~6感染可上调ORF26转录水平;胞外表达的HIV- 1 Tat与HHV~6组合不仅可显著促进ORF26 mRNA转录(t检验，P<0.05)，而且可诱导HHv-8 KS.l蛋白的表达。上述研究结果表明，HHv-6病毒颗粒可诱导HH丫8裂解周期复制，Hlv- 1 Tat蛋白可协同HHv-6增强这种激活作用。提示HHv-6和Hl v-- ITat通过诱导KSHv复制导致病毒数量增加，进而参与KS致病机制。更多还原

【Abstract】 To study the effect of human herpesvirus 6 (HHV-6) and human immunodeficiency virus type 1 (HIV-1) Tat in combination on lytic cycle replication of human herpesvirus 8 (HHV-8), we evaluated HHV-8 ORF26 mRNA in BCBL-1 cells fused with HHV-6-infected cells and cocultured with HHV-6-infected cells by direct cell-cell contact or in Transwell system. RT-PCR and real-time quantitative PCR were performed to detect ORF26 mRNA expression in heterokaryons that were created by BCBL-1 cells fused with HHV-6-infected JJhan cells (also known as HV6-J). The results demonstrated that HHV-8 was significantly activated by HHV-6 in heterokaryons (t test, P < 0.05). Meanwhile, real-time quantitative PCR were carried out to detect ORF26 mRNA expression in BCBL-1 cells cocultured HV6-J by direct cell-cell contact and cultured in the supernatants from HV6-J. And IFN-y and IL-10 expression in their supernatants were monitored by ELISA. The results indicated that ORF26 mRNA in BCBL-1 cells cocultured HV6-J by direct cell-cell contact and cultured in the supernatants from HV6-J were significantly increased compared to that of control (t test, P < 0.05).However, there were no differences in expression of IFN-y and 1L-10 between test and control groups, but the expression of IFN- y in BCBL-1 cultured in the supernatants from HV6-J was increased compared to that of control (t test, P > 0.05). After infection with HHV-6 particles and heat-inactivated or LJV-irradiated viral particles, ORF26 mRNA expression in BCBL-1 cells were elevated on different time point. Furthermore, gene transfection assay and Transwell system were performed to study the effect of extracellular Tat cooperated with HHV-6 and intracellular Tat alone on the expression of ORF26 in BCBL-1 cells. The results from real-time quantitative PCR analysis for ORF26 mRNA showed that HHV-6 and HIV-1 Tat in combination are responsible for HHV-8 replication and induction of lytic cycle proteins (K8.1), but, neither HHV-6 nor extracelluar Tat alone can activate KSHV in Transwell system. Meanwhile, intracellular Tat also failed to induce KSHV lytic phase replication. These findings suggest that both HHV-6 and HIV-1 Tat participate in KS by promoting KSHV replication and, hence, increasing KSHV viral load.更多还原