CV-A16对长爪沙鼠肌肉细胞的杀伤机理研究  

Effect of Coxsackievirus A16 on primary gerbil muscle cells and its mechanism

作　　者：孙一晟[1] 姚苹苹[1] 杨章女[1] 徐芳[1] 卢杭景 伍立志[1] 朱函坪[1] SUN YiSheng;YAO PingPing;YANG ZhangNyu;XU Fang;LU HangJing;WU Lizhi;ZHU HanPing(Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou,Zhejiang 310051,China)

机构地区：[1]浙江省疾病预防控制中心,浙江杭州310051

出　　处：《预防医学》2018年第4期325-328,333,共5页CHINA PREVENTIVE MEDICINE JOURNAL

基　　金：浙江省自然科学基金(LY18H190003);浙江省基础公益技术研究计划(LGF18H100002);浙江省重点研发计划(2018C03070);省部共建重点项目(WKJ-ZJ-1816)

摘　　要：目的研究柯萨奇病毒A组16型(CV-A16)对沙鼠原代肌肉细胞的杀伤机理,为阐明CV-A16的致病机理及沙鼠模型的进一步应用提供依据。方法采用胰蛋白酶/胶原酶双酶法构建沙鼠原代肌肉细胞模型,用不同感染剂量CV-A16感染细胞24h,采用CCK-8法检测细胞存活率,Hoechst 33258染色法检测细胞染色质变化,Annexin V/PI双染法检测细胞凋亡比例,蛋白免疫印迹法检测Caspases家族蛋白、NF-κB通路相关蛋白及JNK激酶表达。结果感染复数(MOI)=0.50和1.00组细胞存活率分别为88.95%和64.05%,与阴性对照组(100.00%)比较,差异均有统计学意义(P<0.05),细胞存活率与MOI呈负相关(rs=-0.857,P=0.014);MOI=0.01、0.10和1.00组细胞凋亡比例分别为7.2%、21.8%和50.7%,细胞凋亡比例与MOI呈正相关(rs=1.000,P<0.001)。CV-A16感染后,Caspase-3和Caspase-8蛋白均被剪切激活,NF-κB通路相关蛋白IκBα、p65和p-p65表达均减少,JNK蛋白磷酸化水平增加。结论 CV-A16可能通过抑制NF-κB通路和激活JNK激酶诱导沙鼠原代肌肉细胞凋亡。Objective To explore the killing mechanism induced by Coxsackievirus A16(CV-A16)in primary muscle cells of gerbils,and to lay the foundations for elucidation the pathogenesis of CV-A16 and the further application of gerbil model.Methods The primary muscle cell model was established by digestion of trypsase/collagenase double enzyme hydrolysis.Primary muscle cells were infected by different dose of CV-A16 and the cell viability was detected by CCK-8 assays.Chromatin condensation and break were measured by Hoechst 33258 staining.The early and last stage of apoptosis cells were measured by AnnexinV/PI double staining.Expression changes of Caspase-3,Caspase-8,JNK and NF-κB pathway proteins were detected by Western Blot.Results The cell viability were 88.95%and 64.05%at groups of different multiplicity of infection(MOI=0.50 and 1.00),which was significantly different from those of the negative control group.The cell viability and multiplicity of infection were negative correlation(rs=-0.857,P=0.014).The apoptosis rates were 7.2%,21.8%and 50.7%at MOI=0.01,0.10 and 1.00 groups,respectively.The apoptosis rate and MOI were positive correlation(rs=1.000,P<0.001).When the primary cells were infected by CV-A16,cleavage of Caspase-3 and Caspase-8 were detected.Western Blot assays showed that the expression of NF-κB pathway proteins IκBα,p65 and p-p65 were reduced,which was different in enterovirus 71-infected cells.The JNK kinase was actived.Conclusion CV-A16 could induce apoptosis in primary muscle cells from gerbils.

关 键 词：柯萨奇病毒 细胞凋亡 沙鼠 机理 

分 类 号：R373.2[医药卫生—病原生物学]