小干扰RNA在心肌细胞中对柯萨奇病毒B3复制的抑制作用研究

目的 通过在原代心肌细胞培养上研究小干扰RNA(small interfering RNA,siRNA)介导的基因干扰对病毒增殖的抑制和细胞内免疫清除作用,评价siRNA在治疗病毒性感染中的可能性.方法 体外制备BALB/c乳鼠心肌细胞,利用脂质体和电穿孔转染方法转染siRNA后感染病毒,流式细胞术、中性红染色和病毒致细胞病变作用(CPE)保护实验评价转染效率和细胞生长状态,空斑形成减少实验、RT-PCR等方法检测抑制病毒程度.结果 通过在HeLa细胞上的筛选,针对病毒不同区域的siRNA呈现出不同的抗病毒作用,靶序列位于2B区的siRNA-3753能显著抑制柯萨奇病毒B3(CVB3)感染.在原代培养的心肌细胞中,转染siRNA-3753后同样显示出很好的抗病毒效果,心肌的搏动、心肌细胞的生长状况保持着较好的状态.而病毒对照和非特异性siRNA-non感染24 h后,细胞停止搏动,逐步变圆,皱缩死亡.测定培养上清和细胞内的病毒滴度,电转siRNA-3753对病毒抑制率可以达到98.1%,脂质体转染siRNA-3753对病毒抑制率为78.2%.电穿孔转染效率可以达到56.03%,脂质体为9.0%.结论 针对CVB3基因组2B区的siRNA在保护心肌细胞抵抗CVB3病毒感染实验中具有抑制病毒复制作用.

Inhibition of Coxsackievirus replication by small interfering RNA in cardiac myocytes

Objectlve To investigate the inhibition of Coxsackievirus B3(CVB3)infection in cardiac myocytes cultured by small interfering RNA(siRNA)-mediated RNA interference and to evaluate the feasibility of siRNA as the prophylaxis and therapy for CVB3 infection.Methods Cardiac myocytes were prepared in vitro and infected with CVB3,and transfected with siRNA by lipofectamin and electroporation.The numbers of beating cardiac myocytes were counted under the microscope.Neutral red staining was used to evaluate the mortality of cardiac myocytes.Antiviral activities of these siRNAs were estimated by observing cytopathic effect(CPE),plaque reduction assay,Western blot assay and RT-PCR.Results siRNA-3753,which aimed at sequence in 2B section of CVB3 genome,displayed a stronger inhibition of CVB3 infection through screening in HeLa cells,siRNA-3753,chosen to transfect cultured neonatal mice cardiac myocytes,Wag observed to keep a good states of growing and beating at 24 h after CVB3 infection.Whereas the cytopathic signature of controlled cells became stopping beating,round and finally the cell fell off the culture plate.The results showed that siRNA-3753 could protect cells significantly,98.1%inhibition of CVB3 replication with electroporation transfection and 78.2%inhibition of CVB3 with liposome transfection.Transfection efficiencies were 56.0 3%and 9.0%by electroporation and lipofectamin,respectively.Conclusion siRNA,which aims at sequence in 2B section of CVB3 genome,can inhibit CVB3 infection in cultured cardiac myocytes.