反义硫代寡核苷酸体外抑制柯萨奇病毒B_3增殖的研究  

Study on the inhibitory effect of antisense phosphorothioate oligodeoxynucleotide on coxsackie virus B_3 replication in vitro

作　　者：齐秀英[1] 李晓眠[2] 刘民[2] 李梅[2] 张国际[2] 

机构地区：[1]天津医科大学流行病学教研究,300070 [2]天津医科大学微生物学教研室,300070

出　　处：《中华实验和临床病毒学杂志》2000年第3期253-256,共4页Chinese Journal of Experimental and Clinical Virology

摘　　要：目的　在CVB3 易感的Vero细胞系中观察与CVB3RNA 5’NCR的nt 5 81- 6 0 1区域互补的 2 1聚硫代AODN抗病毒活性。方法　采用MTT法测定细胞活性 ,直接观察CPE ,测定培养上清TCID50 ,并分别用ELISA和斑点杂交法检测CVB3 抗原及其RNA。结果　 1.AODN可推迟和减轻CPE ,且随AODN浓度增加 ,对CPE的抑制作用增强 ,感染细胞存活率也随AODN浓度升高而升高 ;2 .AODN可抑制CVB3 抗原表达及RNA的合成 ,且呈剂量依赖关系 ;3.培养上清中病毒滴度随AODN浓度升高而降低。AODN抗病毒活性在转染后 48h效果较好 ;10 μmol/L浓度抑制效果较好。本研究结果也显示 10 μmol/LSODN对CVB3 感染细胞CPE有较弱的抑制作用 ,但RODN未显示抗病毒活性 ;AODN未显现对HSV - 1感染细胞CPE抑制活性 ,但对其他肠道病毒如CVB5,Polio - 1和ECHO - 6有较弱的抑制活性。Objective To investigate the antiviral effect of antisense phosphorothioate oligodeoxynucleotide (AODN) on CVB 3 replication in vitro. Methods In this study, 21mer antisense phosphorothioate oligodeoxynucleotide which complemented to nt 581-601 in 5'NCR of Coxsackie Virus B 3 (CVB 3) RNA was used to investigate antiviral activity in Vero cells as a specific inhibitor of CVB 3 replication. Specific AODN of CVB 3 RNA, sense oligodeoxynucleotide (SODN) and randomized nonsense-sequence RODN were synthesized for effect comparison among them. Vero cells infected by CVB 3 were transfected with different concentrations of AODN mediated by Lipofectamine regeant, so that the cells could take in more AODN. Cell control and viral control were set up. The inhibitory effect of AODN on CVB 3 replication was evaluated with a number of variables, including inhibitory rates of cytopathic effect (CPE), cell survival rates by MTT assay, inhibitory rates of CVB 3 antigen by ELISA, inhibitory rates of RNA by dot blotting and 50% tissue culture infective dose (TCID 5). Results The specific AODN could significantly inhibit CPE of CVB 3-infected Vero cells, decrease the production of antigen and RNA of CVB 3 and viral titres, and increase cell survival rates, in a dose-dependent manner. The strongest inhibitory effect appeared at 48 hours after transfection, the most effective concentration of AODN was 10 μmol/L. On the other hand, 10 μmol/L SODN also showed weaker inhibitory effect on CPE of CVB 3-infected cells, but no antiviral effect of 10 μmol/L RODN was shown. AODN showed no-inhibition on HSV-1 replication, but inhibited some entroviruses such as CVB 5, Polio-1 and Echo-6 to some extent. Conclusions These results indicate that nt581-601 in 5'NCR of CVB 3 RNA may play an important role in regulating CVB 3 replication and AODN may have inhibitory effect on CVB 3 replication.

关 键 词：反义寡核苷酸 柯萨奇病毒B3 VERO细胞 

分 类 号：R373[医药卫生—病原生物学]