慢病毒介导绿色荧光蛋白基因转染豚鼠巩膜成纤维细胞的实验研究  

Expression of green fluorescent protein in guinea pig fibroblasts mediated by lentiviral vector

作　　者：张兰兰[1] 于健[2] 吴炳义[1] 刘琼[2] 汤明芳[2] 谭青[2] 胡小娜[2] 

机构地区：[1]南方医科大学南方医院临床医学实验中心,广东省广州市510515 [2]南方医科大学南方医院眼科,广东省广州市510515

出　　处：《眼科新进展》2011年第12期1107-1110,共4页Recent Advances in Ophthalmology

基　　金：国家自然科学基金资助(编号:30901650);南方医院院长基金资助(编号:2008C002)~~

摘　　要：目的观察慢病毒载体转染豚鼠巩膜成纤维细胞的有效性及对细胞活性的影响。方法转染组用慢病毒携带绿色荧光蛋白(lentiviral mediated green fluorescent protein,Lenti-GFP)转染豚鼠巩膜成纤维细胞,对照组加入空白培养液,转染组按最佳感染复数(multiplicity of infection,MOI)为50、100、200、400加入Lenti-GFP,转染2d、3d、4d、5d、6d和7d后,在倒置显微镜下观察绿色荧光蛋白表达情况,流式细胞仪检测转染效率,MTT法检测病毒对细胞增殖的影响。结果 Lenti-GFP转染细胞后48h可以看见绿色荧光;在同一MOI值下,随时间延长转染效率增高,第5天达到高峰;在MOI为400,转染5d时转染效率达82.86%;转染5d行MTT检测显示:MOI为50～400时,同一转染时间各组IOD值与对照组比较,差异均无统计学意义(均为P>0.05)。结论慢病毒载体可以在体外稳定而有效地转染豚鼠巩膜成纤维细胞,且不影响细胞活性,是巩膜成纤维细胞理想的基因转染载体。Objective To investigate the transfection efficiency and activity of cells after lentiviral vector in the guinea pig sclera fibroblasts（GSF） in vitro.Methods The cells were exposed to different concentrations of lentiviral mediated green fluorescent proteins（Lenti-GFP） in transfection groups and the same quality of blank culture medium in control groups.At different multiplicity of infection（MOI;50,100,200,400） and various time points（2 days,3 days,4 days,5 days,6 days and 7 days） after infection,we observed the expression of GFP by inverted fluorescence microscope and flow cytomtery,and calculated the transfection efficiency.We also observed the influence of cells＇ proliferation by Methyl thiazolyl tetrazolium.Results As lenti-GFP transfected cells for 48 hours,the green fluorescence could be seen clearly.At the same MOI value,the transfection efficiency increased with time,and went to peak at the 5th day.When the MOI was 400,the transfection efficiency was 82.86% after five days.When MOI was 50 to 400,the influence of the lentivirus on cells proliferation had no difference compared with the control group by Methyl thiazolyl tetrazolium（P0.05）.Conclusion The lentivirus can be steady and efficient to transfect GSF.It has no influence on cell activity,and will be an ideal gene transfer vector for sclera fibroblasts.

关 键 词：慢病毒载体 绿色荧光蛋白 巩膜成纤维细胞 基因转染 

分 类 号：R772.3[医药卫生—眼科]