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预防医学  2018, Vol. 30 Issue (4): 325-328    DOI: 10.19485/j.cnki.issn2096-5087.2018.04.001
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CV-A16对长爪沙鼠肌肉细胞的杀伤机理研究
孙一晟,姚苹苹,杨章女,徐芳,卢杭景,伍立志,朱函坪
浙江省疾病预防控制中心,浙江 杭州 310051
Effect of Coxsackievirus A16 on primary gerbil muscle cells and its mechanism
SUN Yi-Sheng,YAO Ping-Ping,YANG Zhang-Nyu,XU Fang,LU Hang-Jing,WU Li-zhi,ZHU Han-Ping
Zhejiang Provincial Center for Disease Control and Prevention,Hangzhou,Zhejiang 310051,China
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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24 h,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。
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孙一晟
姚苹苹
杨章女
徐芳
卢杭景
伍立志
朱函坪
关键词 柯萨奇病毒细胞凋亡沙鼠机理    
AbstractObjective 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.
Key wordsCoxsackievirus A16    Apoptosis    Gerbil    Molecular mechanism
     出版日期: 2018-03-26
ZTFLH:  R373.2  
基金资助: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)
通信作者: 朱函坪,E-mail:hanpingzhu@aliyun.com   
作者简介: 孙一晟,博士,主管技师,主要从事病原微生物检测及感染机理研究工作
引用本文:   
孙一晟,姚苹苹,杨章女,徐芳,卢杭景,伍立志,朱函坪. CV-A16对长爪沙鼠肌肉细胞的杀伤机理研究[J]. 预防医学, 2018, 30(4): 325-328.
SUN Yi-Sheng,YAO Ping-Ping,YANG Zhang-Nyu,XU Fang,LU Hang-Jing,WU Li-zhi,ZHU Han-Ping. Effect of Coxsackievirus A16 on primary gerbil muscle cells and its mechanism. Preventive Medicine, 2018, 30(4): 325-328.
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http://www.zjyfyxzz.com/CN/10.19485/j.cnki.issn2096-5087.2018.04.001      或      http://www.zjyfyxzz.com/CN/Y2018/V30/I4/325
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