人偏肺病毒感染人肺上皮细胞后Toll样受体的表达及其功能

摘要

Objective To investigate the expression changes of Toll-like receptors(TLR) and the signaling pathway function on airway epithelial cells infected with human metapneumovirus(hMPV), and to explore the mechanisms of hMPV-induced airway inflammation. Methods A549 cells were infected with hMPV live or co-cultured with UV-inactivated hMPV in vitro. Growth curve of the virus in A549 cells was established and mRNA expression of TLR were detected by RT-PCR assay and real-time PCR assay at 3, 6, 9, 12, 24 h post infection. IFN-α and TNF-α in the culture supernatants were detected by ELISA. Results hMPV was able to replicate in A549 cell, growing to peak titer of 10~(5.2) TCID_(50)/ml at 3 d after infection. Most of TLR mRNA levels were up-regulated by hMPV infection but not UV-inactivated viruses via RT-PCR assay (P<0.05) after 6 h of stimulation. Real-time PCR assay showed that TLR3-4, TLR7-9 mRNA increased after being infected by hMPV in a time-dependent manner. The expression of IFN-α and TNF-α in the cul-ture supernatants were significantly up-regulated after 24 h infected by hMPV. Conclusion hMPV infection up-regulates the expression of TLR in lung epithelial cells. The inflammatory response to hMPV may associ-ate with part of TLR signal transduction pathway.%目的 观察人偏肺病毒(human metapneumoviras)感染人肺上皮细胞后Toll样受体(TLR)表达变化及其信号通路的功能,探讨hMPV诱导气道炎症的部分机制.方法 hMPV感染体外培养的人肺上皮细胞株A549,检测病毒在A549细胞中的生长曲线,并通过RT-PCR,real-timeRT-PCR方法检测细胞TLR mRNA的表达,ELISA检测细胞培养上清IFN-α及TNF-α的表达.结果 (1)hMPV可在A549细胞中复制,感染后3 d达高峰10~(5.2)TCID_(50)/ml.(2)RT-PCR结果提示:hMPV感染A549细胞6 h后大部分TLR的表达均上调.(3)定量PCR结果提示:hMPV感染A549细胞后TLR3-4、TLR7-9的表达增高,且有时间依赖性,而紫外灭活的hMPV刺激细胞后TLR表达无明显变化.(4)ELISA结果提示hMPV感染后24 h,IFN-α及TNF-α的表达均明显升高.结论 人偏肺病毒感染A549细胞后可上调TLR表达,其诱导的炎性反应与部分TLR介导的信号转导途径有关.