【作者】 陈晓红；

【导师】 方峰；

【作者基本信息】 华中科技大学 ， 儿科学， 2011， 博士

【摘要】 [目的](1)研究BML-111对人巨细胞病毒(human cytomegalovirus, HCMV)感染THP-1源巨噬细胞的免疫调节作用及其机制；(2)观察BML-111对HCMV在THP-1源巨噬细胞及人胚肺成纤维细胞(human embryonic lung fibroblast, HEL)中复制增殖的影响。[方法](1) HCMV感染THP-1源巨噬细胞模型制备及实验分组：用100nmol/LPMA诱导THP-1细胞分化为THP-1源巨噬细胞,再用HCMVAD169毒株感染THP-1源巨噬细胞(MOI=0.5)建模,设立模拟感染组、HCMV感染组及HCMV+BML-111组,BML-111的终浓度为100nmol/L;(2)BML-111对炎症因子蛋白表达的影响：以病毒吸附后更换维持液的完成时间为计时起点,在感染后Oh、1h、2h、4h、12h、24h、48h和72h收集各组细胞培养上清液,用双抗体夹心ELISA法检测上清中IL-1β、TNF-α、IL-10和TGF-β表达水平;(3)BML-111对炎症因子mRNA表达的影响：在上述时间点收集各组细胞,用SYBR green real-time RT-PCR法检测THP-1源巨噬细胞中IL-1β、TNF-α、IL-10及TGF-βmRNA表达强度；(4)BML-111对NF-κB p65亚基核转位的影响：用Western blot法于感染后4h检测各组THP-1源巨噬细胞核中NF-κB p65亚基蛋白的表达；(5)BML-111对HCMV在THP-1源巨噬细胞中复制的影响：于HCMV感染(MOI=0.5)THP-1源巨噬细胞后Oh、1h.2h.4h、12h、24h、36h、48h和72h,用SYBRgreen real-time RT-PCR法分别检测各组细胞中IE86和pp65mRNA的水平；(6)BML-111对HCMV在HEL细胞中增殖的影响：HCMV感染HEL细胞(MOI=0.1),设立HCMV感染组与HCMV+BML-111组,逐日光镜观察HEL细胞病变并计数其比率；蚀斑法测定HEL细胞内感染性HCMV滴度。[结果](1)用PMA诱导THP-1细胞48h后,由悬浮生长变成贴壁生长,显示出巨噬细胞形态特征,已分化为THP-1源巨噬细胞；(2)BML-111对炎症因子蛋白表达的影响：与模拟感染组相比,HCMV感染组和HCMV+BML-111组各细胞因子在感染后表达明显升高；与HCMV感染组相比,HCMV+BML-111组IL-1β和TNF-α显著降低,IL-10无差异,TGF-β显著升高；(3)BML-111对炎症因子mRNA表达的影响：与模拟感染组相比,HCMV感染组和HCMV+BML-111组各细胞因子mRNA在病毒感染后明显升高；与HCMV感染组相比,HCMV+BML-111组各细胞因子：mRNA均明显降低；(4)BML-111对NF-κB p65亚基核转位的影响：与模拟感染组相比,HCMV感染组和HCMV+BML-111组细胞核内NF-κB p65亚基蛋白浓度明显升高;与HCMV感染组相比,HCMV+BML-111组核内NF-κB p65亚基蛋白浓度明显降低；(5)BML-111对HCMV在THP-1源巨噬细胞中复制的影响：HCMV感染组和HCMV+BML-111组IE86mRNA及pp65mRNA在感染后表达明显升高；与HCMV感染组相比,HCMV+BML-111组IE86mRNA及pp65mRNA在细胞感染早期(4h内)表达明显升高,但在感染中晚期(24h～72h),pp65mRNA的表达明显降低;(6)BML-111对HCMV在HEL细胞中增殖的影响：与HCMV感染组相比,HCMV+BML-111(?)细胞先1d出现病变,提前2d达到100%细胞病变；感染性病毒滴度提前2d达到峰值,但两组病毒滴度峰值无差异。[结论](1)BML-111对HCMV感染THP-1源巨噬细胞有免疫负调节作用。(2)BML-111对HCMV感染THP-1源巨噬细胞的免疫负调节作用机制可能为：抑制NF-kB的p65亚基核转位,减少促炎因子IL-1β和TNF-α表达；促进抑炎因子TGF-β表达；对抑炎因子IL-10表达无影响；(3)BML-111在感染早期加块HCMV在细胞内的复制速度,在感染晚期抑制pp65基因表达；(4)BML-111在体外对HCMV的感染性病毒增殖量无影响。更多还原

【Abstract】 Objectives:①to investigate the immunomodulatory effect of BML-111on THP-1macrophages during HCMV infection;②to observe the influence of BML-111on HCMV replication in THP-1macrophages and human embryonic lung fibroblasts(HEL)Methods:①a cellular model of THP-1macrophages infected by HCMV was established: THP-1cells were converted into macrophages induced by100nmol/L PMA, and the group division in the experiment was as follows:mock infection,HCMV infection, HCMV+BML-111. HCMV MOI=0.5,the concentration of BML-111was100nmol/L;②the influence of BML-111on the protein expression of inflammatory cytokines was observed:the secreted ILl-β、TNF-α, IL-10and TGF-βin the supernatant of THP-1macrophages medium were Detected by ELISA at0、1h、2h、4h、12h、24h、48h and72h;③the influence of BML-111on the mRNA expression of inflammatory cytokines was observed:test the mRNA levels of ILl-β,TNF-aα, IL-10and TGF-βin the THP-1macrophages were tested by SYBR green real-time RT-PCR at0,1h、2h、4h、12h、24h、48h and72h;④the influence of BML-111on the translocation of NF-κB p65subunit was observed:NF-κB p65in the nucleus of THP-1macrophages Detectede by Western Blot at4h;⑤the influence of BML-111on the HCMV replication in THP-1macrophages was observed:the mRNA levels of IE86and pp65in the THP-1macrophages were tested by SYBR green real-time RT-PCR at0,1h、2h、4h、12h、24h、36h48h and72h;⑥the influence of BML-111on the HCMV replication in HELs was observed:the group division in this part was as follows:HCMV infection and HCMV+BML-111. the patho-morphous changes were observed under microscope,and the infective virus titer changes were examined by plaque assay.Results:①THP-1cells grew in suspension originally, then the cells adhered to the dish bottom and had the morphological characteristics of macrophages after48h of culture with100nmol/LPMA which had been converted into macrophages.②ELISA shows:compared with the group of mock infection, the levels of all the cytokines both in the HCMV infection group and in the HCMV+BML-111group increased significantly; compared with the group of HCMV infection, the levels of lLl-βand TNF-α in the HCMV+BML-111group decreased significantly, the level of TGF-β of the HCMV+BML-111group increased significantly, and there was no differences of the level of IL-10between them.③SYBR green real-time RT-PCR shows:compared with the group of mock infection, the mRNA levels of all the cytokines of the HCMV group and HCMV+BML-111group increased significantly; compared with the group of HCMV infection, the mRNA levels of all the cytokines of the HCMV+BML-111group decreased significantly.④Western Blot shows:compared with the group of mock infection, the level of NF-κB p65in the nucleus of the HCMV group and HCMV+BML-111group increased significantly; compared with the group of HCMV infection, NF-κB p65in the nucleus of the HCMV+BML-111group decreased significantly.⑤SYBR green real-time RT-PCR shows:compared with the group of mock infection, the mRNA levels of IE86and pp65of the HCMV group and HCMV+BML-111group increased significantly; compared with the group of HCMV infection, the mRNA levels of IE86and pp65of the HCMV+BML-111group increased significantly in the early stage after infection, but the mRNA level of pp65decreased significantly in the medium and late stages after infection.⑥Observation under microscope shows:the degree of the patho-morphous of the HCMV+BML-111group reached100%earlier than the group of HCMV infection; and plaque assay shows:the infective virus titer reached the peak earlier than the group of HCMV infection too.Conclusions:①BML-111negatively modulates THP-1macrophages immune response during human cytomegalovirus(HCMV) infection②The negatively modulation of BML-111were related with inhibiting nuclear translocation of NF-κB p65, decreasing the pro-inflammatory cytokines ILl-βand TNF-a,increasing the anti-inflammatory cytokine TGF-β,with no influence on the anti-inflammatory cytokine IL-10.③BML-111accelerates the replication of HCMV in the early stage of infection, but inhibits the expression of pp65gene in the late stage.④BML-111doesn’t increase the peak value of the infective HCMV titer in vitro.更多还原