甲型流感病毒对巨噬细胞的侵染及对干扰素表达的影响

Infection of influenza A virus on macrophages and its effect on interferon expression

目的探讨甲型流感病毒对缺少唾液酸残基巨噬细胞的侵染情况,并初步分析其对IFN表达的影响。方法将PR8、Memphis、Aichi三种甲型流感病毒株以相同的病毒滴度分别侵染MHS肺泡巨噬细胞,对照组(NC)加等量PBS,24 h收集上清及细胞样本。采用RT-PCR法检测实验组病毒的负载量;普通倒置光学显微镜镜下观察实验组及NC细胞病变;实时荧光定量RCR法检测实验组及NC巨噬细胞样本中IFN-β、IFN-γ及Toll样受体3(TLR3)的表达水平,并进行统计学分析。结果MHS巨噬细胞被3株甲型流感病毒株侵染后,镜下均观察到细胞病变,Aichi病变程度最强、Memphis次之、PR8最弱。在细胞和上清样本中检测到Aichi和Memphis核酸拷贝数较高(Aichi:胞内Ct=14.93,上清Ct=19.05;Memphis:胞内Ct=17.15,上清Ct=21.39),PR8拷贝数较低(胞内Ct=26.86,上清Ct=29.31)。与NC比较,3株病毒均诱导IFN-β高表达(Aichi/NC=210、Memphis/NC=93、PR8/NC=33),同时抑制IFN-γ表达,差异均有统计学意义(F=224.00和2637.00,P均<0.001)。此外,Aichi诱导TLR3表达量升高(Aichi/NC=5.03),与NC比较,差异有统计学意义(t=6.40,P=0.031)。结论尽管巨噬细胞表面缺少甲型流感病毒唾液酸受体,但仍可通过其他途径被病毒侵染,并影响其胞内病毒RNA识别受体和IFN的表达,不同毒株之间的侵染能力存在较大差异。

Objective To investigate the infection of influenza A virus(IAV)on macrophages with lack of sialic acid on the cell surface and to explore its influence on the expression of IFN.Methods MHS mouse alveolar macrophage cells were cocultured with IAV(PR8,Memphis,Aichi)at the same titer.The negative control group(NC)was added with equivalent PBS.Supernatant and cell lysate were collected 24 hours after infection.IAV RNA in each sample was amplificated by RT-PCR.The cytopathic effect was observed in IAV groups and NC group under ordinary inverted optical microscope.Expression levels of IFN-β,IFN-γand toll-like receptor3(TLR3)were determined in IAV groups and NC group by real-time RT-PCR and retrospectively analyzed.Results All of three strains of IAV induced positive cytopathic effect on infected microphages with the lesion degree from strong to weak of Aichi,Memphis and PR8.High nucleic acid copy numbers of Aichi and Memphis(Aichi:intracellular Ct=14.93,supernatant Ct=19.05;Memphis:intracellular Ct=17.15,supernatant Ct=21.39)but low copy number of PR8(intracellular Ct=26.86,supernatant Ct=29.31)were detected in infected macrophage cells and supernatant.Compared with NC,it showed the up-regulation of IFN-β(Aichi/NC=210,Memphis/NC=93,PR8/NC=33)and the down-regulation of IFN-γin all of macrophages infected by IAV with statistically significant differences(F=224.00 and 2637.00,P both<0.001).In addition,it presented a significantly higher expression level of TLR3 in macrophages infected by Aichi(Aichi/NC=5.03,t=6.40,P=0.031).Conclusions The macrophages are not susceptible to IAVs due to the lack of sialic acid residues which is considered as influenza virus receptor,but they may be infected in other ways.IAVs can affect cell biological function including intracellular viral RNA recognition receptors and interferon expression.The infection ability to macrophages among different strains is quite different.