RSV感染小胶质细胞活化Toll样受体3、7介导炎性反应的初步研究

Preliminary study of inflammatory effects induced by Toll-like receptor 3 and Toll-like receptor 7 activation in respiratory syncytial virus infected BV2 cells

目的探讨呼吸道合胞病毒(RSV)感染小鼠小胶质细胞(BV2)后,Toll样受体3(TLR3)和Toll样受体7(TLR7)的活化及其介导的炎性反应。方法①RSV体外感染BV2,利用免疫荧光技术分别在12、24和36 h检测细胞内RSV-F蛋白。②分别于感染4、8、12、16、24和36 h收集细胞,以未感染细胞作为正常组。用TRIzol提取细胞总RNA,通过半定量RT-PCR法检测TLR3、TLR7的mRNA转录水平的变化。③Western blot法分别检测感染4、8、12、16、24和36 h后的细胞内核转录因子(NF-κB)蛋白水平的变化。④ELISA法检测细胞上清液中肿瘤坏死因子α(TNF-α)含量的变化。结果①荧光显微镜下可以见到FITC标记RSV-F蛋白的绿色荧光随感染时间的延长而增强。②TLR3、TLR7在感染RSV后mRNA水平在观察的时间内表达均升高并呈时间依赖性关系。③各感染时间点NF-κB蛋白水平表达均升高并呈时间依赖性关系。④感染RSV后,细胞上清液中TNF-α含量均升高并呈时间依赖性关系。结论 RSV感染BV2后可以上调TLR3、TLR7,从而使下游细胞炎性因子TNF-α分泌升高,过度的炎性作用可能与神经系统症状的发生相关。

Objective To investigate the affect of respiratory syncytial virus （RSV） infected mouse glioma cells （BV2） by activation of Toll-like receptor 3 （ TLR3 ） and Toll-like receptor 7 （ TLR7 ）, which induced the down- stream inflammatory factors excessive in the nervous system. Methods （1） The infection of RSV in BV2 cells were confirmed by immunofluorescence using F proteins antibody at 12, 24 and 36 h post infection. （2） The quantitative of TLR3, TLR7 mRNA were performed total RNA extracted by TRIzol at 4, 8,12, 16, 24 and 36 h post infection using RT-PCR. The uninfected cells were also harvested at same time as control. （3） The expression levels of nucle- ar factor-kappa B（NF-KB）were detected by Western blot at 4, 8, 12, 16, 24 and 36 h post infection. The unin- fected cells were also harvested at same time as control. （4） The concentration of tumor necrosis factor-alpha（ TNF- α） in cell supernatant was detected by ELISA at 4, 8, 12, 16, 24 and 36 h post infection. The uninfected cells were also harvested at same time as control. Results （1） The green fluorescent signals were gradually increased from 12 h to 36 h post infection. （2） The expression levels of TLR3, TLR7 mRNA increased along with the exten- sion of infection. （3） The expression levels of NF-KB also increased along with the extension of infection. （4） The concentration of TNF-α in cell supernatant was elevated along with the extension of infection. Conclusion RSV in- fection in BV2 cells could induce inflammatory cytokines, such as TNF-α secretion by activation of TLR3, TLR7. The excessive expression of inflammatory cytokines may be associated with nervous system diseases.