TLR7在呼吸道合胞病毒感染A549细胞诱导Ⅰ型干扰素中的作用

The role of TLR7 in A549 cells with the respiratory syncytial virus infection induced type Ⅰ interferon

目的探讨Toll样受体7(TLR7)在呼吸道合胞病毒(RSV)感染A549细胞诱导产生I型干扰素(IFN)抗病毒免疫反应中的作用。方法实验分正常对照组、RSV感染组、TLR7 siRNA沉默组,分别于感染的4、8、12、24 h后收集各组细胞以及培养上清液。TLR7 siRNA通过瞬时转染A549细胞,半定量RT-PCR法检测TLR7基因沉默效果;TRIzol试剂提取细胞总RNA,检测TLR7、干扰素调节因子7(IRF7)、IFN-α、IFN-β的mRNA表达量变化;通过Western blot法检测TLR7蛋白表达量;ELISA法检测感染不同时间点A549细胞培养上清液中IFNα/β含量的变化。结果 1转染24h后TLR7 siRNA-2有效抑制TLR7 mRNA表达;2 RSV感染A549细胞后,TLR7、IRF7、IFN-α/β的mRNA以及TLR7蛋白的表达量均升高,并与RSV感染之间存在时间依赖性关系;沉默TLR7后,TLR7、IRF7、IFN-α/β的mRNA以及TLR7蛋白的表达量较感染组均有明显下降;3 RSV感染A549细胞后,明显上调细胞培养上清液中IFN-α/β的水平,TLR7siRNA沉默组IFN-α/β的表达水平较RSV感染组均下降,差异有统计学意义,且IFN-α的表达量下调更为显著。结论RSV感染A549细胞后,TLR7被活化能够诱导产生I型IFN,发挥抗病毒作用,且TLR7诱导产生的I型IFN以IFN-α为主。

Objective This experiment was designed to explore the antiviral effect of TLR7 when A549 cells were infected by respiratory syncytial virus（ RSV） to induce the production of type I interferon（ IFN）. Methods This experiment was divided into normal control group,RSV infection group and TLR7 silence group. In each group cell and culture supernatant were collected after having been infected for 4,8,12 and 24 h. TLR7 siRNA was transfected in A549 cells by transient transfection. With the use of the RT-PCR assay,TLR7 gene silencing effect could be detected; with the use of Trizol reagent,the total RNA could be extracted and the expression dynamics of mRNA in TLR7,IRF7,IFN-α and IFN-β could be detected; with the use of Western blot,protein expression could be tested; with the use of ELISA,the variation of IFNα / β in the culture supernatant of A549 cells infected in the different points of time could be detected. Results 1 24 h after transfection,TLR7 siRNA-2 could restrain the expression of TLR7 mRNA effectively with statistical significance. 2 The expression of mRNA in TLR7,IRF7 and IFN-α / β,together with TLR7 protein,which existed time dependent relation with RSV infection,had increased after A549 cells had been infected by RSV. Meanwhile,compared with RSV infection group,the expression of mRNA in TLR7,IRF7 and IFN-α / β,together with TLR7 protein had decreased obviously in TLR7 silence group. 3Compared with RSV infection group,the expression of IFN-α / β in cell culture supernatant and in TLR7 silent group had decreased,and the expression of IFN-α decreased obviously. It went without saying that the difference of decrease in two groups had statistical significance. Conclusion After cell A549 have been infected by RSV,TLR7 could be activated and then induce type I interferon after having been infected by RSV,which play the role in anti-virus. Noticeably,IFN-α accounts for the most of e type I interferon.