【摘要】 目的探讨甲型流感病毒H1N1/FM1感染小鼠巨噬细胞不同时间点NOD样受体P3(NLRP3)炎症小体信号通路的变化。方法以小鼠单核巨噬细胞系RAW264.7为研究对象,甲型流感病毒H1N1/FM1感染1周作为实验组,感染6 h作为对照组,ELISA检测细胞培养上清中IL-1β的浓度,荧光定量PCR检测炎症小体信号通路中NLRP3、caspase-1、IL-1β的mRNA相对表达量,蛋白免疫印迹法检测NLRP3、caspase-1、cleaved-caspase-1的蛋白相对表达量,免疫荧光染色法定性检测NLRP3的蛋白表达。结果细胞培养上清液中IL-1β的浓度实验组高于对照组(P<0.01),其NLRP3 mRNA和蛋白的表达量低于对照组降低(P均<0.05),caspase-1、IL-βmRNA相对表达量以及caspase-1、cleaved-caspase-1蛋白相对表达量均高于对照组(P均<0.05)。免疫荧光染色中实验组NLRP3蛋白荧光强度弱于对照组。结论甲型流感病毒H1N1/FM1感染小鼠巨噬细胞可以激活NLRP3炎症小体信号通路,促进IL-1β的分泌,且在感染后期(约1周)有更强的激活效应。更多还原

【Abstract】 Objective To investigate the variation of NOD-like receptor P3(NLRP3) inflammosome signaling pathway at different time points in mouse macrophages infected by influenza A virus H1N1/FM1.Methods Mouse monocyte-macrophage cell line RAW264.7 was used in this experiment.In the study group,the cells were infected with H1N1/FM1 for 1 week,and those were infected for 6 h in the control group.The expression of IL-1β in the cell supernatant was measured by ELISA.The relative expression of NLRP3,caspase-1 and IL-1β mRNA was investigated by fluorescent quantitative PCR.The expression levels of NLRP3,caspase-1 and cleaved caspase-1 proteins were quantitatively detected by Western blot.The expression of NLRP3 protein was observed by immunofluorescent staining.Results In the study group,the expression of IL-1βin the cell supernatant was significantly up-regulated(P < 0.01),the expression levels of NLRP3 mRNA and protein were significantly down-regulated(both P < 0.05),the relative expression of caspase-1 and IL-β mRNA,and caspase-1 and cleaved-caspase-1 proteins were significantly up-regulated(all P < 0.05) compared with those in the control group.The fluorescent intensity of NLRP3 protein in the study group was lower than that in the control group.Conclusions Infection of macrophage cells with influenza A virus H1N1/FM1 can activate the NLRP3 inflammosome signaling pathway and promote the secretion of IL-1β.Futhermore,the activating effect can be enhanced during the late stage of infection(approximately 1 week).更多还原