呼吸道合胞病毒感染巨噬细胞诱导炎性基因表达的部分机制研究

目的探讨呼吸道合胞病毒(RSV)感染巨噬细胞时前炎介质肿瘤坏死因子-α(TNF-α)和诱导型一氧化氮合酶(iNOS)的基因表达变化及其调控的相关机制,为研究RSV的致病机制及有效预防和治疗RSV疾病提供新的思路。方法以RSV感染RAW264.7巨噬细胞,并设立不同的感染时间点(1h、4h、8h、16h和24h),同时给予PDTC(核转录因子NF-κB的特异性抑制剂)处理。以紫外线灭活RSV(UV-RSV)来分析有传染性的病毒的感染变化。收集各组细胞,用Western blot法检测细胞核内活性NF-κBp65蛋白的表达,半定量RT-PCR法检测TNF-α和iNOS mRNA表达量。结果RSV感染4h后,细胞核内活性NF-κBp65蛋白、TNF-α和iNOS mRNA表达均明显升高,各指标的变化与正常对照相比,差异均有显著性,并且与RSV感染存在时间依赖关系。当加入PDTC抑制NF-κB的入核活化后,则可显著下调相应时间点的RSV感染升高的TNF-α和iNOS mRNA表达量,使其降低至基线水平。而UV-RSV感染后并不引起NF-κB蛋白、TNF-α和iNOS mRNA的表达量增加(P>0.05)。结论RSV感染巨噬细胞可诱导前炎基因TNF-α和iNOS的大量表达,其表达可能主要依赖NF-κB活化,并且与病毒复制有关。提示在RSV感染的巨噬细胞中,NF-κB活化对TNF-α和iNOS基因表达具有重要的正调控作用。

Study on the partial mechanisms of inflammatory gene expression induced by respiratory syncytial virus infection in macrophages

To investigate the related regulatory mechanisms of production of proinflammatory mediators such as tumor necrosis factor-alpha(TNF-α)and inducible nitric oxide synthase(iNOS)in macrophages(RAW264.7 cells)infected with respiratory syncytial virus(RSV)in order to investigate the pathogenesis and to provide the beneficial idea for the clinical therapy of this infection,the RAW264.7 cells infected with RSV were treated or untreated with nuclear factor κB(NF-κB)inhibitor pyrrolidine dithiocarbamate(PDTC)to inhibit the activation of NF-κB.In addition,RSV was ultraviolet light inactivated(UV-RSV)and was analyzed for infectious virus.At the indicated time points of RSV infection(1,4,8,16 and 24 h),the macrophages were collected and the expression of active NF-κB p65 protein in cell nuclei protein,as an indicator of NF-κB activation,was detected by Western blot assay.The expression of TNF-α and iNOS mRNA were evaluated semiquantitatively by RT-PCR,respectively.It was found that RSV infection could markedly up-regulate the expression of active NF-κB protein since 4 hours postinfection in a time-dependent manner,and those of TNF-α and iNOS mRNA levels also rose in infected cells.The changes of each index varied significantly compared with the control group.Whereas PDTC inhibitor treatment could significantly down-regulate the activation of NF-κB,and the expression of TNF-α and iNOS mRNA were also decreased accordingly since 4 hours after infection.However,UV-RSV treatment did not induce obviously the increase of NF-κB activation,TNF-α and iNOS mRNA expression.These current data indicate that RSV infection could induce apparent increase of proinflammatory genes of TNF-α and iNOS in macrophages,which is mainly NF-κB-dependent and viral replication,suggesting that the activation of NF-κB may play an important role in the positive regulation of TNF-α and iNOS expression in RSV-infected macrophages.