小檗胺影响牛病毒性腹泻病毒体外复制的研究  

Study on the Effect of Berbamine on in vitro Replication of Bovine Viral Diarrhea Virus

作　　者：付强[1] 李泽宇 贺渊秀 塞力克·杰恩斯 李丹[1] 葛丽娟 李金月 谭美玲 杨莉 岳剑波 史慧君[1] FU Qiang;LI Zeyu;HE Yuanxiu;Sailike·Jieensi;LI Dan;GE Lijuan;LI Jinyue;TAN Meiling;YANG Li;YUE Jianbo;SHI Huijun(College of Veterinary Medicine,Xinjiang Agricultural University,Urumqi 830052,China;Department of Biomedical Science,City University of Hong Kong,Hong Kong 999077,China)

机构地区：[1]新疆农业大学动物医学学院,乌鲁木齐830052 [2]香港城市大学生物医学系,中国香港999077

出　　处：《中国动物传染病学报》2023年第4期33-40,共8页Chinese Journal of Animal Infectious Diseases

基　　金：自治区天山青年计划项目青年博士科技人才培养项目(2018Q069);自治区高校科研计划项目(XJEDU2018Y019);自治区创新环境(人才、基地)建设专项-天山创新团队(2020D14005);新疆农业大学畜牧学博士后流动站工作(168138、168134)。

摘　　要：本研究旨在研究天然小分子药物小檗胺(BBM)是否影响牛病毒性腹泻病毒(BVDV)体外复制。分别配制0、1、5、8、10、15、20μmol/L浓度的BBM,使用MTT法检测BBM对MDBK细胞增殖的影响;并选择抑制作用相对较小浓度的BBM处理牛肾细胞(MDBK)8 h后感染BVDV,使用免疫荧光技术检测BVDV双链RNA(ds RNA)含量,确定最佳用药浓度;分别使用实时荧光定量PCR、Reed-Muench法、倒置显微镜观察来检测BBM对BVDV RNA复制、病毒滴度变化、致细胞病变效应(CPE)的影响。结果显示:10μmol/L BBM对BVDV dsRNA积累具有显著性抑制作用,同时该浓度对MDBK细胞的毒性作用较小;同时采取荧光定量PCR检测和滴度测定,发现在72 h内BVDV感染BBM处理的MDBK细胞与对照相比,5’UTR mRNA水平和子代病毒滴度显著性降低,在BVDV感染24 h和36 h时,子代病毒滴度差异性最大;BVDV感染对照组处理的MBDK细胞会造成严重的CPE现象,而BVDV感染BBM处理的细胞CPE现象较弱。以上结果表明,本研究初步验证BBM显著抑制BVDV在细胞中的复制。This study was conducted to investigate whether small molecule drug Berbamine(BBM) affected Bovine viral diarrhea virus(BVDV) replication in vitro.Preparations of BBM at 0,1,5,8,10,15 and 20 μmol/L concentrations were added to MDBK cell cultures and the cells were infected with BVDV 8 h later.MTT assay was performed to detect the effect of BBM on the proliferation of MDBK cells and optimal concentration.The accumulation of BVDV double strand RNA(dsRNA) was tested in BBM treated MDBK cells in immunofluorescence staining.At the same time,quantitative real-time PCR and Reed-Muench method inverted microscope were applied to determine the effect of BBM on BVDV RNA replication,viral titer alteration and CPE.The results showed as low as 10 μmol/L of BBM significantly inhibited the accumulation of BVDV dsRNA,and the cell toxicity was small at this concentration.Quantitative realtime PCR and titer assay showed that the level of 5'UTR mRNA and viral titer in MDBK cells treated with BBM were significantly lower than control at 72 h post inflection and the most difference of virus titers between BBM treated or non-treated cells was presented at 24 h and 36 h post infection.In addition,BVDV infection caused severe CPE in non-treated MBDK cells but less CPE in BBM-treated cells.In conclusion,BBM treatment significantly inhibited BVDV replication in cells.

关 键 词：小檗胺 牛病毒性腹泻病毒 病毒复制 双链RNA 

分 类 号：S859.7[农业科学—临床兽医学]