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目的:通过将筛选的UL29shRNA表达质粒与抗病毒药物阿昔洛韦(ACV)的抗病毒效果及细胞毒性进行比较,探讨RNA干扰在细胞水平上对HSV-2病毒的抑制效果。方法:将筛选的干扰效果最好的表达质粒UL29-shRNA1641作为RNA干扰组与抗病毒药ACV进行对HSV-2的抑制效果的比较研究,分为RNAi组、ACV组和RNAi+ACV组。采用实时荧光定量PCR检测UL29mRNA的相对表达量,Karber法检测细胞上清液中病毒滴度的变化、蛋白印迹法(Western blot)检测ICP8的相对表达量,对RNAi与ACV抑制效果进行比较,通过WST-1(Water-Soluble Tetrazolium-1)法对质粒转染复合物(质粒+转染试剂)和ACV的细胞毒性进行比较。结果:RT-PCR检测三组mRNA的相对表达水平,其中RNAi组UL29基因相对表达量高于ACV组(P<0.05);RNAi+ACV组UL29基因相对表达量明显低于RNAi组和ACV组(P<0.05)。Karber法检测三组细胞上清液病毒滴度表明,RNAi组病毒滴度高于ACV组(P<0.05),RNAi+ACV组的病变病毒滴度明显低于RNAi组和ACV组(P<0.05)。Western blot检测ICP8相对表达量,ACV组的ICP8(UL29编码的单链DNA结合蛋白,主要作用是在HSV-2 DNA复制过程中与复制叉产生了单链DNA结合,防止其重新配对形成ds DNA或被核酸酶降解)相对表达量比RNAi组低(P<0.05)。RNAi+ACV组ICP8相对表达量降低最为明显,与ACV组和RNAi组相比有显著差异(P<0.05)。WST-1法对转染复合物与ACV的细胞毒性进行比较,其中RNAi中质粒和转染试剂对细胞的毒性明显小于ACV。结论:在RNAi与ACV对HSV-2抑制效果的比较中,ACV要好于RNAi,两者联合使用的抑制效果好于单独使用RNAi或ACV。在抑制效果都较好的情况下比较,RNAi中使用的质粒与转染试剂对细胞的影响比ACV小。
OBJECTIVE: To investigate the inhibitory effect of RNA interference on HSV-2 virus at the cellular level by comparing the antiviral effect and cytotoxicity of the screened UL29 shRNA expression plasmid with antiviral drug acyclovir (ACV). Methods: The screening of the best interference expression plasmid UL29-shRNA1641 RNA interference group and the antiviral agent ACV on HSV-2 compared the inhibitory effect, divided into RNAi group, ACV group and RNAi + ACV group. The relative expression of UL29 mRNA was detected by real-time fluorescence quantitative PCR. Karber’s method was used to detect the change of virus titer in the cell supernatant. The relative expression of ICP8 was detected by Western blot and the inhibitory effect of RNAi and ACV was compared. WST-1 (Water-Soluble Tetrazolium-1) method was used to compare the cytotoxicity of plasmid transfection complex (plasmid + transfection reagent) and ACV. The relative expression of UL29 gene in RNAi group was higher than that in ACV group (P <0.05). The relative expression level of UL29 gene in RNAi + ACV group was significantly lower than that in RNAi group and ACV group P <0.05). The titer of virus supernatant in three groups by Karber assay showed that the titer of virus RNAi group was higher than that of ACV group (P <0.05), and the titer of virus in RNAi + ACV group was significantly lower than that of RNAi group and ACV group ). The relative expression of ICP8 was detected by Western blot. ICP8 of ACV group (UL29-encoded single-stranded DNA binding protein, the main role is in the replication of HSV-2 DNA replication fork produced a single-stranded DNA binding to prevent its re-pairing to form dsDNA Or degraded by nucleases) relative expression amount was lower (P <0.05) than RNAi group. Compared with ACV group and RNAi group, RNAi + ACV group showed the most significant reduction in ICP8 expression (P <0.05). WST-1 method was used to compare the cytotoxicity of the transfection complex with ACV, in which the RNAi plasmid and transfection reagent were significantly less cytotoxic than ACV. CONCLUSION: ACV is better than RNAi in the inhibition of HSV-2 by RNAi and ACV. The combined effect of RNAi and ACV is better than RNAi or ACV alone. The plasmids and transfection reagents used in RNAi had less effect on cells than ACV when the inhibition was better.