流感病毒诱导小鼠胶质细胞的细胞因子级联反应

Cytokine cascades in mouse glia cells induced by influenza virus infection

目的:探讨流感病毒感染星形胶质细胞后释放的细胞因子是否会诱导正常胶质细胞趋化因子及促炎细胞因子转录水平的变化,从而产生细胞因子级联效应。方法:从新生小鼠大脑皮质分离培养神经胶质细胞,并进一步纯化星形胶质细胞,经纯度鉴定后,用感染复数为2的流感病毒H1N1和H3N2进行体外感染星形胶质细胞,分别于6小时和24小时收获上清,采用超滤分子截留的方法,去除流感病毒颗粒。用不同时间点的条件上清,分别刺激星形胶质细胞和小胶质细胞,24小时后提取RNA并进行反转录,利用Real-Time PCR检测促炎因子TNF-α、IL-1β、IL-6,趋化因子IP-10、MCP-1、MIP-1转录水平的变化。结果:不同时间点的条件上清皆可诱导正常胶质细胞的促炎症因子TNF-α、IL-1β、IL-6和趋化因子IP-10、MCP-1、MIP-1的转录水平发生不同程度的上调,产生细胞因子级联效应。结论:流感病毒H1N1和H3N2感染星形胶质细胞后所引起的细胞因子风暴,可诱导正常胶质细胞的细胞因子转录水平显著上调,引发细胞因子级联反应,其可能与流感病毒感染中枢神经系统所引发的细胞因子风暴及免疫病理损伤存在一定关系。

Objective：To investigated whether the cytokines secreted from influenza virus-infected mouse astrocyte cells could induce the changes of transcription levels in chemokines and proinflammatory cytokines in mouse microglia and astrocytes, thus resulting in cytokine cascades. Methods：The mixed gila cells isolated from neonatal mice cerebral cortex were cultured and further astrocytes were purified. The primary mouse astrocytes were infected by H1N1 or H3N2 influenza viruses in a multiplicity of infection（ MOI）2 in vitro. The supernatant were collected at 6 hours and 24 hours post infection, then the viral particles were removed by ultrafihration molecules retain technique. The conditioned supernatant of different time point was used to cultivate the primary mouse microglia and astrocytes for 24 hours. The RNA were extracted to detect the transcriptional levels of proinflammatory cytokines TNF-α, IL-1 β, IL-6 and chemokines IP-10,MCP-1, by Real-Time PCR. Results：The transcription levels of proinflammatory cytokines TNF-α, IL-1 β, IL-6 and chemokines IP-10, MCP-1, MIP-1 were up regulated in astrocytes and microglia which cultivated with conditioned supernatant. Conclusion：The cytokine cocktail generated by influenza virus-infected astrocyte cells could increase the transcription levels of cytokines in normal glia cells significantly, result in cytokine cascades, perhaps it has relationship with cytokine storm and immunopathogenic injury of CNS induced by influenza virus infection.