质粒载体介导的短发夹RNA对RPE细胞转染效率及毒性研究  

Kinetics study of plasma vector carrying shRNA transfection of green fluorescent protein gene and cytotoxicity to RPE cells in vitro

作　　者：蔡春梅 范思均 梁歌 姜严明 冯婧 CAI Chun-mei;FAN Si-jun;LIANG Ge;JIANG Yan-ming;FENC Jing(Department of Ophthalmology,the Roeket Army General Hospital,Beijing 100088,China)

机构地区：[1]火箭军总医院眼科,北京100088

出　　处：《解剖科学进展》2018年第6期614-617,共4页Progress of Anatomical Sciences

基　　金：2016年度全军医学科技青年培育项目(16QNP033)

摘　　要：目的连接针对人血管内皮生长因子(VEGF)设计的短发夹RNA与质粒载体,并转染视网膜色素上皮(RPE)细胞,观察其转染效率及对细胞的毒性。方法设计针对人VEGF的短发夹RNA(shRNAs,V1,V2),V3为阴性对照即不含特异性shRNA,其表达载体为pGCSilencer^(TM)/U6/Neo/GFP/VEGF RNAi。用Lipofectamine^(TM) 2000转染RPE细胞,用倒置荧光显微镜及流式细胞仪观察其转染效率;用MTT法观察其对细胞的毒性。结果质粒载体介导的GFP在细胞中的表达呈黄绿色,弥漫于整个胞浆和胞核,转染后24h GFP开始表达,转染后48h表达明显增强。流式细胞仪检测质粒pGCSilencer^(TM)/U6/Neo/GFP/VEGF RNAi对体外培养的RPE细胞转染效率为43.9%,质粒转染后MTT法测定细胞活性的OD值与对照组相比明显降低(P=0.004)。结论质粒pGCSilencerTM/U6/Neo/GFP/VEGF RNAi可以有效地转染体外培养的RPE细胞,但质粒转染对细胞有明显的毒性。Objective To design short hairpin RNA(shRNA) targeting human vascular endothelial growth factor(VEGF)ligated to plasma vector of pGCSilencer^TM/U6/Neo/GFP, and evaluate the efficiency of transfection and cytotoxicity to human retinal pigment epithelium(RPE) cells in vitro. Methods shRNAs(V1,V2) specific for human VEGF were designed. DNA expression vector is pGCSilencer^TM/U6/Neo/GFP. V3 is negative control nonspecific shRNA ligated by JiKai company(China). RPE cells were transfected with plasma by LipofectamineTM 2000, the transfection efficiency of plasma to RPE cells was evaluated by flow cytometer and fluorescent microscope. The effect of plasma vector on RPE cells’ growth was determined by methyl thiazoly tetrazolium(MTT) assay. Results Twenty-four hours after transfection, RPE cells emitted green fluorescence under the fluorescence microscope. GFP expression in RPE cells could be detected 24 h after transfection, and yellow-green fluorescence of GFP gene appeared in the cytoplasm and nuclear. The expression level of GFP gene increased 48 h, the transfection efficiency of pGCSilencerTM/U6/Neo/GFP/VEGF RNAi was 43.9% measured by flow cytometer. The OD records were lower in transfected group than in untransfected group(P=0.004). Conclusion RPE cells can be transfected effectively by plasma vector pGCSilencerTM/U6/Neo/GFP/VEGF RNAi, with cytotoxicity to RPE cells transfected with plasma vector.

关 键 词：质粒载体 视网膜色素上皮细胞(RPE) 细胞毒性 

分 类 号：Q782[生物学—分子生物学]