呼吸道合胞病毒感染后气道上皮细胞Toll样受体4的表达及其功能

Toll-like receptor 4 expression and function of respiratory syncytial virus-infected airway epithelial cells

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TLR1-10 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 3h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TLR4 mRNA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。

Objective 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 TLR1-10 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 analyzed by 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 value = 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 epithelial 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.