RNA干扰技术抑制哺乳动物细胞绿色荧光蛋白表达的研究

DOWNREGULATION OF ENHANCED GREEN FLUORESCENCE PROTEIN GENE EXPRESSION BY RNA INTERFERENCE IN MAMMALIAN CELLS

目的本研究旨在探讨阳离子脂质体的体外转染效率、化学合成双链RNA(dsRNA)诱导RNA干扰(RNAi)的效应强度及其作用特点,为采用RNAi技术开展基因治疗提供实验依据。方法①采用磷酸钙沉淀法构建293T/GFP细胞株。②体外化学合成dsRNA,经由三种不同的阳离子脂质体TransIT-TKO,Oligofectamine reagent,Lipofectamine2000导入293T/GFP细胞,通过荧光显微镜、流式细胞仪测定荧光强度的变化,观察不同阳离子脂质体的体外转染效率。③采用dsRNA-Lipofectamine2000复合物转染293T/GFP细胞。dsRNA分设5个不同的浓度(0.01,0.02,0.04,0.08,0.16μmol/L)转染细胞48h以观察剂量效应关系。dsRNA0.08μmol/L/Lipofectamine20008μl转染细胞,于4个不同的时间点(12h,24h,48h,72h)观察时间效应关系。结果绿色荧光蛋白序列特异性dsRNA(dsRNA-eGFP)经由三种不同的阳离子脂质体导入细胞均可产生RNAi效应,序列非特异性dsRNA(dsRNA-unrelated)无抑制效应。在三种不同的阳离子脂质体中,Lipofectamine2000的转染效率最强,转染48h可将293T/GFP细胞荧光强度降低约80%,与空白载体组比较差异显著,TransIT-TKO组,Oligofectamine reagent组分别降低41.02%、37.45%,与空白载体组比较无显著性差异,而且TransIT-TKO、Oligofectamine的细胞毒性较强。结果还表明dsRNA/Lipo-fectamine2000诱导的RNAi效应具有剂量及时间双重依赖性,dsRNA0.08μmol/L/Lipofectamine20008μl/6孔培养板转染细胞48h的抑制效应最强。结论①体外化学合成的dsRNA可有效诱导哺乳动物细胞出现序列特异性基因沉默效应。②三种不同的阳离子脂质体中Lipofectamine2000的转染效率最强,且细胞毒性轻微。③dsRNA/Lipofectamine2000诱导的RNAi效应具有剂量及时间双重依赖性。

Objective RNA interference （RNAi） is a recently observed process by which double-stranded RNA （dsRNA） directs sequence-specific degradation of messenger RNA （mRNA） in animal and plant cells. The aim of this study was to investigate the efficiency and mechanism of RNAi induced by chemically synthetic small interference RNA （siRNA） in mammalian cells. Methods We transduced 293T cells by enhanced green fluorescence protein （EGFP） gene via lentivirus and obtained EGFP positive cells i. e. 293T/GFP cells. Then we introduced chemically synthetic 21-nucleotide siRNA duplexes into 293T/GFP cells by means of TransIT-TKO, Oligofectamine reagent, and Lipefectamine 2000 respectively. The effects of gene silencing were observed by both fluorescence microscopy and flowcytometry. To study whether the silencing effect was processed in a dose and time-dependent manner, we transfected the 293 T/GFP cells by means of Lipefectamine 2000 with 0.01, 0.02, 0.04, 0.08, 0. 16 p.mol/L dsRNA-EGFP for a 48 h exposure, or transfected the cells with 0. 08 μmol/L dsRNA-EGFP at four preselected time points i.e. 12 h, 24 h, 48 h, 72 h. Results EGFP expression was significantly and specifically inhibited by the corresponding dsRNA, but not by unrelated dsRNA. In three different vectors, Lipefectamine 2 0 0 0 demonstrated the highest transfection efficiency with a 48 h exposure. The decrease in EGFP fluorescence intensity was approximate 80%. Although TransIT-TKO and Oligofectamine displayed similar trends, the transfections were inefficient （41.02% for TranslT-TKO, 37.45% for Ohgofectamine） and often toxic. The results also exhibited that siRNAinhibited the EGFP gene expression in a dose and time-dependent manner. Conclusion The Lipofectamine 2000 was a better transfection reagent for RNAi. RNAi induced by chemically synthetic siRNA processed in a time and dose dependent manner, and hence, RNAi pathway seems operative in mammalian embryo cells. RNAi may be developed into a potential tool for gene therapy.