瑞德西韦对感染肠道病毒71型的人横纹肌瘤细胞和ICR乳鼠的抗病毒活性  

Antiviral activity of remdesivir against human rhabdomyosarcoma cells and ICR suckling mice infected with enterovirus 71

作　　者：任晓风 闫赟政 李微 李月香 肖军海 曹瑞源 李永刚 REN Xiaofeng;YAN Yunzheng;LI Wei;LI Yuexiang;XIAO Junhai;CAO Ruiyuan;LI Yonggang(Department of Pathogenic Biology,School of Basic Medical Sciences,Jinzhou Medical University,Jinzhou 121000,China;Institute of Pharmacology and Toxicology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)

机构地区：[1]锦州医科大学基础医学院病原生物学教研室,辽宁锦州121000 [2]军事科学院军事医学研究院毒物药物研究所,北京100850

出　　处：《吉林大学学报（医学版）》2021年第6期1446-1454,共9页Journal of Jilin University：Medicine Edition

基　　金：辽宁省教育厅基础研究项目(JYTJCZR201909)。

摘　　要：目的:探讨瑞德西韦(RDV)在细胞与动物水平对肠道病毒71型(EV71)的抗病毒活性,并阐明其抗病毒作用机制。方法:基于人横纹肌瘤(RD)细胞进行RDV抗肠道病毒活性评价,检测RDV对EV71、柯萨奇病毒A6型(CA6)、肠道病毒D68型(EVD68)和柯萨奇病毒16型(CA16)的半数毒性浓度(CC50)和半数有效浓度(EC50),计算选择指数(SI)。将RD细胞分为细胞对照组(不处理)、病毒对照组和给药组。病毒对照组RD细胞给予EV71病毒液,给药组RD细胞再给予不同浓度(0.005、0.015、0.046、0.137、0.410、1.230、3.700、11.110、33.330和100.000μmol·L^(-1))RDV。72 h后,采用CellTiter-Glo?Luminescent检测试剂盒测定各组RD细胞活性。在抗EV71细胞活性评价实验中,将RD细胞分为给药组和病毒对照组,给药组RD细胞给予不同浓度(0.03、0.10、0.30、0.80和2.50μmol·L^(-1))RDV。30 h后,采用实时荧光定量PCR(RTqPCR)法检测各组RD细胞中EV71 RNA表达水平,采用Western blotting法和免疫荧光法检测各组RD细胞中EV71结构蛋白VP1表达量。采用加药时序实验验证RDV的抗病毒作用阶段。在抗EV71动物药效学实验中,将35只ICR乳鼠随机分为安慰剂组(给予2%Tween 80,n=12)、3.0 mg·kg^(-1)RDV组(给予3.0 mg·kg^(-1)RDV,n=12)和1.5 mg·kg^(-1)组RDV组(给予1.5 mg·kg^(-1)RDV,n=11),每只乳鼠经腹腔攻毒5×103PFU。4 h后进行第1次给药,连续给药2周,观察并记录乳鼠生存率。在攻毒后第3天采用RT-qPCR法检测各组乳鼠各种组织中病毒载量(即EV71 RNA拷贝数)。结果:RDV对EV71、CA6、EVD68和CA16的EC50分别为(0.05±0.01)、(0.14±0.06)、(0.02±0.01)和(0.10±0.03)μmol·L^(-1)。与病毒对照组比较,0.10、0.30、0.80和2.50μmol·L^(-1)RDV组RD细胞中EV71 RNA表达水平降低(P<0.05或P<0.01),0.80和2.50μmol·L^(-1)RDV组RD细胞中EV71结构蛋白VP1表达量降低。与病毒对照组比较,0.80μmol·L^(-1)RDV组RD细胞中Ⅲ和Ⅳ阶段(病毒复制阶段)时EV71病毒基因组RNA拷贝数明显�Objective:To explore the antiviral activity of remdesivir(RDV)against enterovirus 71(EV71)in the cellular and animal levels,and to clarify its antiviral mechanism.Methods:The antienterovirus activity of RDV was evaluated based on the human rhabdomyosarcoma(RD)cells.The half toxic concentration(CC50)and half effective concentration(EC50)of RDV for EV71,Coxackie virus 6(CA6),enterovirus 68(EVD68),and Coxackie virus 16(CA16)were detected,and the selection index(SI)was calculated.The RD cells were divided into cell control group,virus control group(without treatment)and administration groups.The RD cells in virus control group were given EV71,and the RD cells in administration groups were given different concentrations(0.005,0.015,0.046,0.137,0.410,1.230,3.700,11.110,33.330,and 100.000μmol·L^(-1))of RDV.After 72 h,CellTiter-Glo?Luminesent assay kit was used to determine the activities of RD cells in various groups.In the cell activity evaluation experiment of anti-EV71,the RD cells were divided into administration groups and virus control group.The RD cells in administration groups were given different concentrations(0.30,0.10,0.30,0.80 and2.50μmol·L^(-1))of RDV.After 30 h,the expression levels of EV71 RNA in the RD cells in various groups were detected by Real-time fluorescence quantitative PCR(RT-qPCR)method,and the expression levels of EV71 structural protein VP1 in various groups were detected by Western blotting and immunofluorescence methods.Time of addition assay was used to conform the antiviral stage of RDV.In anti-EV71 animal pharmacodynamics,35 ICR suckling mice were randomly divided into placebo group(given 2%Tween 80,n=12),3.0 mg·kg^(-1)RDV group(given 3.0 mg·kg^(-1)RDV,n=12)and1.5 mg·kg^(-1)RDV group(given 1.5 mg·kg^(-1)RDV,n=11);each suckling mouse was challenged intraperitoneally with 5×10^(3)PFU.The first dose was given after 4 h,and the treatment lasted for 2 weeks.The survival rates of the sucking mice in various groups were observed and recorded.On the 3 rd day after challenge,the viral l

关 键 词：瑞德西韦 肠道病毒71型 人横纹肌瘤细胞 ICR乳鼠 

分 类 号：R512.5[医药卫生—内科学]