中华皮肤科杂志 ›› 2011, Vol. 44 ›› Issue (11): 772-775.

• 论著 • 上一篇    下一篇

小干扰RNA抑制2型单纯疱疹病毒作用的研究

张云青1,赖维2,谢淑霞3,关蕾4,李欢4,5,李刚1,4   

  1. 1. 中山大学附属第三医院
    2. 广州中山大学附属第三医院皮肤科
    3. 广州中山医科大学第三附属医院
    4.
    5. 中山大学附属第一医院
  • 收稿日期:2011-01-10 修回日期:2011-02-19 出版日期:2011-11-15 发布日期:2011-11-01
  • 通讯作者: 李刚 E-mail:ligangzh@pub.guangzhou.gd.cn

Inhibitory effects of small interference RNA （siRNA） on herpes simplex virus type 2

  • Received:2011-01-10 Revised:2011-02-19 Online:2011-11-15 Published:2011-11-01

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摘要：

目的 研究小干扰RNA（siRNA）在体外抑制2型单纯疱疹病毒（HSV-2）的作用。方法 根据HSV-2 VP16和DNA聚合酶基因序列设计、合成siRNA。采用病毒产量减少实验测定siRNA对病毒复制的抑制，在预防性作用研究中，24孔板Vero细胞转染siRNA，4 h后换液并接种HSV-2病毒（0.02 ～ 0.05 PFU/细胞）；在治疗性作用研究中，24孔板Vero细胞接种HSV-2，1 h后转染siRNA。荧光定量RT-PCR测定siRNA对病毒靶基因mRNA水平的影响。结果 分别针对HSV-2 VP16基因及DNA聚合酶基因设计的siRNA——VP2-1和DP2-1，在预防性及治疗性作用研究中均能有效抑制病毒复制，使病毒滴度降低约1个log10 。siRNA VP2-1使病毒VP16 mRNA的水平在感染后6 h和12 h分别降低58.6％和79.5％，而siRNA DP2-1使病毒DNA多聚酶的mRNA水平分别降低59.8％和77.4％。结论 HSV-2 VP16和DNA聚合酶基因的siRNA体外能有效抑制HSV-2复制，VP16和DNA多聚酶基因可以作为RNA干扰抑制HSV病毒复制的靶基因。

关键词: 小干扰RNA

Abstract:

Objective To investigate the antiviral effect of siRNA on herpes simplex virus type 2 （HSV-2） in vitro. Methods Different siRNA duplexes targeting HSV-2 VP16 and DNA polymerase genes were designed and chemically synthesized. The antiviral effect of siRNA duplexes was evaluated by virus yield reduction assay. In detail, Vero cells cultured in 24-well plates were transfected with different siRNAs 4 hours before （preventive effect experiment） or 1 hour after （therapeutic effect experiment） the inoculation with HSV-2 （0.02 to 0.05 plaque forming unit/cell）. After additional culture for 40 to 48 hours, the expression of target genes in Vero cells was measured by real-time quantitative reverse transcription （RT）-PCR. Results Both the siRNA duplex VP2-1 targeting HSV-2 VP16 and DP2-1 targeting DNA polymerase gene resulted in a remarkable decrease in viral titer by about 1 log10 in both the preventive and therapeutic effect experiment. As the preventive effect experiment showed, the expression of HSV-2 VP16 mRNA was decreased markedly by 58.6％ and 79.5％ by VP2-1, respectively, and that of HSV-2 DNA polymerase mRNA by 59.8％and 77.4％ by DP2-1, respectively, at 6 and 12 hours after the infection. Conclusions The siRNA duplexes targeting HSV-2 VP16 and DNA polymerase genes could inhibit the proliferation of HSV-2. Hence, HSV-2 VP16 and DNA polymerase genes may serve as the target genes of RNA interference to inhibit HSV-2 replication.

Key words: siRNA

中图分类号: 

  • R75