摘要目的 观察呼吸道合胞病毒(RSV)感染后气道上皮细胞Toll样受体4(TLR4)表达变化及其信号通路的功能,探讨RSV诱导气道炎症的机制.方法 体外培养人气管上皮细胞株9HTEo,以RSV感染复数为10感染上皮细胞,用半定量逆转录PCR(RT-PCR)检测TLRl～10 mRNA表达;用定量PCR检测TLR4 mRNA表达的动态变化;用流式细胞术检测TLR4蛋白表达及与细胞凋亡的关系;感染后再用TLR4激动剂脂多糖(LPS)刺激细胞,用酶联免疫法(ELISA)检测上清液中白细胞介素-8(IL-8)含量以观察病毒诱导表达的膜TLR4蛋白功能.RT-PCR和流式细胞术实验分为正常组和RSV感染组,用GraphPad 4.0统计软件进行配对t检验;实时定量PCR实验分为正常组、RSV感染组和紫外线灭活RSV感染组,采用Kruskal-Wallis检验分析;ELISA实验分为正常组、RSV感染组、单独LPS刺激组和RSV-LPS共刺激组,采用One-way ANOVA检验分析.结果 (1)RSV感染组TLR2～10 mRNA表达均上调(t值为3.49～14.47,P均＜0.05),以TLR2和TLR6变化最为显著;定量PCR结果提示:感染组TLR4 mRNA 3 h后开始增高(Kruskal-Wallis检测值=8.82,P＜0.05,n=6),紫外灭活RSV组TLR4 mRNA表达无明显变化;(2)流式细胞术结果表明:RSV感染组膜上TLR4表达比正常组增高(平均荧光强度:1.27±0.48,0.97±0.25,t:2.39,P＞0.05,n=10),感染组胞内TLR4表达比正常组降低(平均荧光强度:3.08±1.38,3.36±1.31;t=2.92,P＞0.05,n=10),感染组膜TLR4阳性细胞中(93.32±1.7)%为膜黏连蛋白-5阳性细胞;(3)RSV-LPS共刺激组培养上清液中IL-8含量明显高于单纯LPS刺激组(F=59.29,P＜0.01,n=3).结论 RSV感染后,上皮细胞TLR4mRNA和蛋白表达水平上调,与TLR4信号通路相关的IL-8分泌增加;TLR4与上皮细胞凋亡的关系提示TLR4及其信号通路可能参与了RSV诱导的上皮细胞急、慢性炎症反应.

abstractsObjective To observe the epithelial Toll like receptor(TLR)4 expression changes and the signaling pathway function after respiratory syncytial virus(RSV)infection and to explore the mechanisms of RSV-induced airway inflammation.Methods 9HTEo-human tracheal epithelial cell line was infected by RSV(MOI=10),and TLRl-10 mRNA were detected by RT-PCR assay at 3 h post RSV infection.TLR4 mRNA was detected by real time Q-PCR assay at 3 h,6 h and 9 h post RSV infection,and TLR4 protein expression and cell apoptosis were determined by flow cytometry at 24 h post RSV infection.IL-8 in supernatant was detected by ELISA after RSV-infected cells exposed to lipopolysaccharide(LPS).A normal control group and a RSV infection group were set up for the RT-PCR and flow cytometry experiments,and the data were analyzed by paried t test using GraphPad 4.0 software.A normal group,a RSV group and a UV-inactivated RSV group were set up for the real time Q-PCR,experiments,and the data were analyzedby Kruskal-Wallis test.The ELISA experiments were divided into 4 groups including a normal control.a RSV,a LPS stimulation,and a RSV plus LPS co-stimulation groups,and the data were analyzed by Oneway ANOVA test.Results (1)TLR2-10 mRNA level was significantly up-regulated(t value of TLR2-10:3.49～14.47,P＜0.05),especially TLR-2,6 enhanced expression,compared with the normal epithelial cells.Real time Q-PCR assay showed that TLR4 mRNA started to increase at 3hr(Kruskal-Wallis test valne=8.82,P＜0.05,n=6)and significantly elevated at 9 hour(Kruskal-Wallis test value=6.62,P＜0.05,n=6).UV inactivated-RSV had no effect on the TLR4 mRNA level.(2)Flow cytometry showed that membrane TLR4 mean fluorescence intensity(MFI)increased(RSV:1.27±0.48,normal:0.97±0.25;t=2.39,P＞0.05,n=10)while cytoplasmic TLR4 MFI simultaneously decreased(RSV:3.08±1.38,normal:3.36±1.31,t:2.92,P=0.225,n=10).Percentage of membrane TLR4-positive cells was higher in RSV infected population[RSV:(11.99±7.74)%,normal:(1.16±0.47)%,Mann-Whitneyt t value=0.001,P＜0.01,n=8],most(93.32±1.7)%of which were Annexin V positive.IL-8 was significantly induced in the RSV plus LPS costimulation group compared with RSV group(F=59.29,P＜0.01,n=3). Conclusions RSV induced epithetial TLR4 up-regulation,localization changes from cytoplasm to membrane.IL-8 secretion through TLR4 signaling pathway and epithelail cell apoptosis in membrane TLR4 positive population.These results indicate TLR4 iS involved in RSV-induced acute or chronic epithelial-dependent inflammation.which might contribute to acute or chronic airway inflammation.