在Hep G2细胞中利用siRNA沉默GFP基因的实验探讨

目的:通过探讨小分子双链RNA(siRNA)通过RNA干扰(RNAi)机制沉默肝癌细胞株Hep G2荧光素报告基因GFP的各项生物学指标,为进一步实验和临床研究提供依据.方法:通过体外实验,对siRNA和siRNA脂质体的稳定性和细胞毒性进行评估;通过荧光标记技术,研究不同时间siRNA脂质体的转染效率;通过荧光显微镜下观察转染细胞和RT-PCR检测靶基因mRNA表达,确定不同种类、形式、剂量的siRNA对靶基因的沉默效能.结果:在空白培养液中,siRNA和siRNA脂质体可稳定至4h,在5%血清中2～4h后明显降解;100nM的siRNA和siRNA脂质体无明显细胞毒性;siRNA脂质体转染细胞株的效率随时间不同而不同,4h可达90%;非特异性siRNA/脂质体无沉默靶基因表达作用,特异性siRNA/脂质体对靶基因的沉默作用在一定范围内随剂量升高(20nM、50nM、100nM)而升高.结论:siRNA的稳定性可满足实验需要,且无明显细胞毒性,特异性siRNA转染肝癌细胞株能有效抑制靶基因表达,用脂质体转染可提高转染效率.siRNA通过RNAi机制沉默靶基因表达,为肿瘤的治疗和基础研究提供了新的工具和思路.

Silence Target GFP Expression in Hep G2 Cell by siRNA

Objective:To research several biological indices of siRNA silencing target green fluorescent protein(GFP) gene expression through RNAi mechanism,provides information for further experimental and clinic research. Methods:Through test in vitro,evaluated the stability and cytotoxicity of siRNA/liposomes;through fluorescent labeling technology with Cy3,investi-gated the trasfection efficiency of siRNA-liposomes;through the observation under fluorescent microscope and RT-PCR with gel electrophoresis technology,assayed the expression of mRNA of target gene,to reveal the silence effect to target gene by siRNA of different type,form and dosage. Results:the siRNA /liposomes maintained stability till 4 hours in culture solution without serum,degraded obviously in 5% serum after 2～4 hours;there was no obvious cytotoxicity by 100nM siR-NA/liposomes;the transfection efficiency of siRNA-liposomes depended on time and reached 90% at 4 hour;non-special siRNA had no silent effect on target gene expression,but special siRNA/li-posomes could silence target gene expression and increased with dosage(20nM,50nM,100nM). Conclusion:The stablity of siRNA can meet the demand of test and there is no obvious cytotoxicity,special siRNA transfect hepatocarcinoma cell can silence target gene expression,the liposomes can improve the efficiency of transfection. siRNA and RNAi provide new tool and thought for therapy of tumor and the basic study.