H6N6亚型禽流感病毒感染RAW264.7细胞炎症模型的建立  

Establishment of an Inflammatory Model of RA W264.7 Cells Infected with H6N6 Subtype Avian Influenza Virus

作　　者：杨鑫[1,2] 王建俊 徐浩 李俊贤 任涛 欧德渊 YANG Xin;WANG Jianjun;XU Hao;LI Junxian;REN Tao;OU Deyuan(College of Animal Science,Guiyang 550025,China;Tongren Center for Prevention and Control of Animal disease,Tongren 554300,China)

机构地区：[1]贵州大学动物科学学院,贵阳550025 [2]铜仁市动物疫病预防控制中心,铜仁554300

出　　处：《经济动物学报》2024年第1期18-24,共7页Journal of Economic Animal

基　　金：贵州省科技计划项目[黔科合基础(2020)1Y136,黔科合支撑(2017)2534]。

摘　　要：探讨H6N6亚型禽流感病毒的感染量、感染时间与被感染的RAW264.7细胞炎性因子水平变化的相关性,为建立RAW264.7细胞体外炎症模型提供参考。根据Reed-Muench法计算病毒滴度(TCID_(50)),以100 TCID_(50)的H6N6亚型禽流感病毒感染RAW264.7细胞2 h,弃病毒液,加入含5%FBS的DMEM培养液继续培养,分别于12,24,48 h收集上清液或细胞,CCK8测定细胞活力;透射电子显微镜检测细胞超微结构;ELISA检测小鼠白介素-1β(IL-1β)、白介素-2(IL-2)、白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的含量;qRT-PCR检测IL-1β、IL-2、IL-4、IL-6和TNF-αm RNA表达水平。结果表明:H6N6感染各时间段细胞活力显著下降(P<0.01);细胞中线粒体肿胀,自噬体增多;IL-1β、IL-2、IL-6和TNF-α含量显著升高(P<0.01);IL-1β、IL-2、IL-4、IL-6和TNF-αmRNA表达水平显著升高(P<0.01),感染24 h最高。成功构建了H6N6亚型禽流感病毒体外感染RAW264.7细胞模型,为进一步研究其致病机制提供参考。The correlation between infection amount and time of H6N6 subtype avian influenza and the dynamic changes of inflammatory level in infected RAW264.7 cells were investigated in the study to provide a reference for establishing an inflammatory model of RAW264.7 cells in vitro.The virus titer(TCIDso)was calculated by Reed-Muench method.RAW264.7 cells were infected with 100 TCIDso of H6N6 subtype avian influenza virus for 2 hours.The virus venom was discarded and the culture was continued by adding DMEM medium containing 5%FBS.The culture supernatant or cells were collected at 12,24 and 48 h,respectively,and the cell viability was determined by CCK8.The ultrastructure of cells was observed by transmission electron microscope.The levels of interleukin-1β(IL-1β),interleukin-2(IL-2),interleukin-6(IL-6)and tumor necrosis factor-α(TNF-α)in mice were detected by ELISA.The mRNA expression levels of IL-1β,IL-2,IL-4,IL-6 and TNF-αwere detected by qRT-PCR.The results showed that the cell viability decreased significantly after H6N6 infection(P<0.01),mitochondria were swollen,autophagosomes were increased,and the contents of IL-1β,IL-2,IL-6 and TNF-αwere significantly increased(P<0.01),the mRNA expression levels of IL-1β,IL-2,IL-4,IL-6 and TNF-αwere significantly increased(P<0.01),and reached the peak at 24 h after infection.The results showed that the RAW264.7 cell model infected with H6N6 subtype avian influenza virus in vitro was successfully constructed,which would provide conditions for further study of its pathogenic mechanism.

关 键 词：H6N6亚型禽流感病毒 RAW264.7细胞 炎症 模型 

分 类 号：S852.65[农业科学—基础兽医学]