CAJ | 학술논문

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.
목적 통과재원대심기세포배양상연구소간우RNA(small interfering RNA,siRNA)개도적기인간우대병독증식적억제화세포내면역청제작용,평개siRNA재치료병독성감염중적가능성.방법 체외제비BALB/c유서심기세포,이용지질체화전천공전염방법전염siRNA후감염병독,류식세포술、중성홍염색화병독치세포병변작용(CPE)보호실험평개전염효솔화세포생장상태,공반형성감소실험、RT-PCR등방법검측억제병독정도.결과 통과재HeLa세포상적사선,침대병독불동구역적siRNA정현출불동적항병독작용,파서렬위우2B구적siRNA-3753능현저억제가살기병독B3(CVB3)감염.재원대배양적심기세포중,전염siRNA-3753후동양현시출흔호적항병독효과,심기적박동、심기세포적생장상황보지착교호적상태.이병독대조화비특이성siRNA-non감염24 h후,세포정지박동,축보변원,추축사망.측정배양상청화세포내적병독적도,전전siRNA-3753대병독억제솔가이체도98.1%,지질체전염siRNA-3753대병독억제솔위78.2%.전천공전염효솔가이체도56.03%,지질체위9.0%.결론 침대CVB3기인조2B구적siRNA재보호심기세포저항CVB3병독감염실험중구유억제병독복제작용.
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.