鸡冠状病毒感染Vero细胞后NLRP3炎症小体信号通路的活化

Activation of NLRP3 Inflammasome Signaling Pathway in Vero Cells Infected with Infectious Bronchitis Virus

目的探讨鸡冠状病毒[即传染性支气管炎病毒(IBV)]感染Vero细胞后,NLRP3炎症小体的变化。方法通过荧光染色、酶联免疫吸附实验、聚合酶链式反应(PCR)方法检测IBV Beaudette株感染非洲绿猴肾细胞Vero细胞不同时间点NLRP3炎症小体相关的信号分子。结果IBV感染Vero细胞后,病毒开始增殖并有明显的细胞病变效应(CPE)现象;通过荧光染色和流式细胞术检测胞内活性氧水平,发现IBV可使Vero细胞内活性氧含量随感染时间延长而增加;酶联免疫吸附实验检测细胞上清液中IL-1β与IL-18的分泌量,发现IBV能促进细胞IL-1β、IL-18的分泌;qRT-PCR检测NLRP3炎症小体相关基因(NLRP3、Caspase-1、IL-1β和IL-18)的转录水平,发现IBV感染Vero细胞后,所有检测基因mRNA表达量均在不同时间点高于对照组。结论IBV感染Vero细胞后可活化NLRP3炎症小体信号通路,并进一步促进IL-1β、IL-18的分泌。

Objective To investigate the changes of NLRP3 inflammasome in African green monkey kidney epithelial cells(Vero cells)infected with avian coronavirus,also known as infectious bronchitis virus(IBV).Methods NLRP3 inflammasome related signaling molecules in IBV Beaudette strain infected Vero cells were detected by fluorescence staining,enzyme-linked immunosorbent assay(ELISA)and quantitative reverse transcription polymerase chain reaction(qRT-PCR)at different time points.Results Virus proliferation and cytopathic effect(CPE)were detected in IBV infected Vero cells.The levels of intracellular reactive oxygen species(ROS)were detected by fluorescence staining and flow cytometry.And it was found that the ROS levels increased with the prolongation of IBV infection.The levels of IL-1βand IL-18 were detected by ELISA.And it was found that IBV infection promoted the secretion of IL-1βand IL-18 in Vero cells.The mRNA expression levels of NLRP3 inflammasome related genes(NLRP3,Caspase-1,IL-1βand IL-18)were detected by qRT-PCR.And it was found that the mRNA expression levels of all detected genes were higher than those of the control group at different time points after IBV infection.Conclusion IBV infection activates the NLRP3 inflammasome signaling pathway and promotes the secretion of IL-1βand IL-18 in Vero cells.