短双链RNA对CVB3病毒细胞内清除作用的可行性研究及其机制探讨

Intracellular dearance of Coxsakievirus B3 infection by short interfering RNA and its mechanism study

目的通过观察短双链RNA（Short interfering RNA，siRNA）体外转染HeLa细胞评价siRNA抗CVB3病毒感染的可行性和抑制作用机制。方法通过细胞病变作用保护实验，选择合适的siRNA剂量。Western Blot、RT．PCR等方法在体外检测siRNA抗CVB3病毒复制作用及作用途径。结果siRNA．3753通过脂质体转染试剂能高效转入HeLa细胞，转染效率可达98．77％，在细胞内可稳定长达48h。siRNA-3753的浓度为0．6gmol／L对病毒抑制率高同时对细胞不会产生毒性作用，该浓度作为本次研究的实验剂量。siRNA-3753抗CVB3病毒感染作用是通过siRNA直接降解病毒基因得到的，而不是通过激活PKR或干扰素等其他未知因素起效的。结论siRNA可以高效、较长时间的转染入HeLa细胞内，在低剂量时即产生较好的抗病毒感染作用，通过直接降解病毒基因组的途径特异性抑制CVB3病毒的复制及感染。

Objective To evaluate the possibility of short interfering RNA （siRNA） inhibiting Coxsackievirus B3 （CVB3） infection in vitro, and discover the mechanism initially. Methods We obtained proper effective dosage of siRNA by observing cytopathic effect （ CPE ）. Estimate its antiviral activities and its pathway of siRNA by Western Blot assay and RT-PCR. Results Results showed that siRNA-3753 can be effectively transfected into HeLa cells, we can achieve a high transfection efficiency up to 98.77 % and its effect can last for 48 h stably in cells. 0.6 μmol/L siRNA-3753 got a high inhibiting effect of virus and didn＇t show any toxicity to cells. So we consider this concentration as the experimental concentration, siRNA-3753 can debase virus reproduction. The antiviral effect is sequence-specific and is not attributable to either interferon or the interferon response effectors protein kinase R （PKR）. Conclusion The data confirmed that siRNA can effectively inhibit CVB3 infection in vitro, its antivirus effect was gained from specific debase of virus genome.