慢病毒载体介导增强型绿色荧光蛋白转导兔角膜上皮细胞的体外实验研究

Lentivirus vector-mediated enhanced green fluorescence protein transfection on in vitro corneal epithelial cells of rabbits

目的观察慢病毒载体(lentiviral vectors,LVs)用于兔角膜上皮细胞基因转染的有效性。方法兔角膜上皮细胞的原代及传代培养并做细胞鉴定;慢病毒载体介导的增强型绿色荧光蛋白(lenti-EGFP)以不同的感染复数(multiplicity of infection,MOI=0、1、10、50、100、500)转染实验细胞,寻找最佳转染剂量;于转染后24、48、72、96h运用倒置荧光显微镜观察不同MOI下增强型绿色荧光蛋白(enhanced green fluorescence protein,EGFP)的表达并计算细胞转染率;RT-PCR方法检测EGFP基因的表达情况。结果 EGFP于转染48h即开始有表达,随着转染时间的延长其表达增强。MOI=1、10、50、100时,角膜上皮细胞转染率随着感染复数的增加而增加,分别为(4.5±0.6)%、(30.4±0.9)%、(51.9±1.4)%、(75.4±0.7)%,各组间差异显著(P<0.05);MOI在100与500时,转染率差异不显著(P>0.05),即最适感染复数为100。RT-PCR结果提示转染细胞组EGFP基因有表达。结论慢病毒载体能够有效转染离体兔角膜上皮细胞。

Objective To observe the effect of lentivirus vector-medicated enhanced green fluorescence protein （EGFP） transfection on in vitro corneal epithelial cells.Methods Primary corneal epithelial cells of rabbits were cultured and transfected with lentivirus vector into different multiplicity of infection （MOI） in order to find the best transfection dose.After 24,48,72,and 96 h of transfection,expression of EGFP was observed under microscope and detected by RT-PCR.The transfection rate of corneal epithelial cells was calculated in different MOI.Results EGFP was expressed in corneal epithelial cells 48 h after transfection,which increased with the increasing transfection time.The transfection rate of corneal epithelial cells was 4.5%±0.6%,30.4%±0.9%,51.9%±1.4%,and 75.4%±0.7%,respectively,when MOI was 1,10,50,and 100（P0.05）.However,no difference was found in the transfection rate of corneal epithelial cells when MOI was 100 and 500.The optimol MOI was 100.RT-PCR showed that EGFP gene was expressed in transfected corneal epithelial cells.Conclusion Lentivirus vector can effectively transfect corneal epithelial cells in vitro.