脂质体介导寡脱氧核糖核苷酸转染人视网膜色素上皮细胞的转染率及分布测定

Detection of transfection efficiency and distribution of oligodeoxynuclotide into human retinal pigment epithelial cells mediated by cationic liposome

目的:观察阳离子脂质体LipofectamineTM 2000介导的寡脱氧核糖核苷酸(ODNs)在体外培养的人视网膜色素上皮(RPE)细胞中的转染效率及其在细胞中的分布。方法:在阳离子脂质体LipofectamineTM 2000的介导下将人工合成的FAM荧光标记的ODNs转入人RPE细胞中,荧光显微镜下观察转染后20 min、1 h、2 h、4 h、6 h、12 h、24 h其在细胞内的分布,流式细胞仪检测其在RPE细胞中的转染效率;并用MTT法检测脂质体处理组、脂质体-ODNs复合物处理组和正常对照组的细胞增生活性,观察脂质体的毒性。结果:LipofectamineTM 2000能迅速将FAM-ODNs转入人RPE细胞的胞质和胞核内,4～6 h达高峰,转染效率高达(85.85±5.75)%,与其他时点比较差异有统计学意义(P<0.05),24 h后其转染率还有(70.11±5.81)%;且脂质体对RPE细胞无明显细胞毒性,3组间细胞增生活性比较,差异无统计学意义(P>0.05)。结论:LipofectamineTM 2000能有效地将FAM-ODNs转入人RPE细胞中,可作为一种安全有效的基因转移载体用于基因治疗。

Aim： To investigate the transfection efficiency and distribution of oligodeoxynuclotide in human retinal pigment epithelial cells mediated by cationic liposome LipofectamineTM 2000.Methods：Fluorescence FAM labeled oligodeoxynuclotide was introduced into RPE cells by LipofectamineTM 2000.The location of fluorescence in RPE cells at 20 min,1 h,2 h,4 h,6 h,12 h,and 24 h after transfection was observed by fluorescence microscope,transfection efficiency was detected by flow cytometry,and the cytotoxicity was measured by MTT assay.Results： FAM-ODNs complexed with LipofectamineTM 2000 were transfected into cytoplasm and nucleus of RPE cells rapidly,and the highest transfect rate reached（85.85±2.75）% after 4 to 6 h incubation（P〈0.05）,（70.11±5.81）% after 24 h.No cytotoxicity was observed（P〉0.05）.Conclusion： LipofectamineTM 2000 could effectively transfect FAM-ODNs into RPE cells and it is a safe and effective vector for gene therapy.