【摘要】 目的:观察呼吸道合胞病毒感染肺上皮细胞A549后Toll样受体3表达的变化,探讨Toll样受体3介导呼吸道合胞病毒感染肺上皮细胞的炎性反应及其介导的信号转导通路。方法:实验于2006-05/12在安徽医科大学基础医学院分子生物学实验室完成。用呼吸道合胞病毒感染体外培养的A549细胞,于感染后4,8,16,24h收集细胞和细胞培养上清。用Trizol提取细胞总RNA,RT-PCR法检测TOLL样受体3mRNA、肿瘤坏死因子αmRNA表达的变化;提取细胞总蛋白和核蛋白,免疫印迹法分别检测TOLL样受体3蛋白、核内活性核因子κB的变化;用ELISA检测细胞培养上清肿瘤坏死因子α的表达。以未感染病毒的细胞为正常对照组。结果:①呼吸道合胞病毒感染A549细胞后,Toll样受体3和肿瘤坏死因子α的mRNA和蛋白的表达量均升高且有时间依赖性,Toll样受体3mRNA24h表达量是基础表达量的5倍多,肿瘤坏死因子αmRNA24h表达量是基础表达量的3倍多。②A549细胞中核内活性核因子κB有基础表达,经由呼吸道合胞病毒感染,核内活性核因子κB随感染时间的延长升高;同时呼吸道合胞病毒感染能促进Toll样受体3蛋白的表达,高于正常对照组(P<0.01)。③呼吸道合胞病毒感染能促进A549细胞分泌肿瘤坏死因子α,感染24h分泌达到高峰。结论:呼吸道合胞病毒感染A549细胞后上调Toll样受体3表达,其诱导的炎性反应与Toll样受体3介导的信号转导途径有关。更多还原

【Abstract】 AIM: To investigate the expression changes of Toll-like receptor 3 (TLR3) on human lung epithelial cells (A549) infected with respiratory syncytial virus (RSV),and study the TLR3 signal transduction pathway and its mediated inflammatory response to RSV. METHODS: The experiment was accomplished in the Department of Microbiology,Anhui Medical University between May and December 2006. A549 cells infected with RSV in vitro were used to collect cells and cellular culture supernatant. Trizol was used to extract total RNA of cells,and RT-PCR was used to evaluate the expression of TLR3 mRNA and tumor necrosis factor (TNF)-α mRNA. After extracting the total protein and nucleoprotein,the expression of TLR 3 protein and nuclear factor (NF)-κB activity was detected by Western-Blot. The expression of TNF-α in culture supernatant was measured ELISA. Whereas those uninfected cells were taken as controls. RESULTS: ①RSV infection to A549 cells increased the amounts of mRNA and protein of TLR 3 and TNF-α in a time-dependent manner. The expression of TLR 3 mRNA was more than 5 times as many as basal expression,that of TNF-α mRNA was more than 3 times.②The basal expression of NF-κB was increased with the prolong of RSV infection to A549 cells; meanwhile,RSV infection promoted the expression of TLR 3 protein,which was higher than that of control group (P < 0.01).③RSV could improve A549 cells secrete TNF-α,and the secretion peaked at 24 hours of infection. CONCLUSION: RSV infection to A549 cells can up-regulate the expression of TLR 3. The inflammatory response to RSV may associate with the TLR3 signal transduction pathway.更多还原