Lenti-eGFP体外转染兔角膜上皮细胞的实验研究  

Lenti-eGFP transfection into rabbit corneal epithelial cells in vitro

作　　者：王继东[1] 马晓辉[2] 兰芬[3] 王健 陈余 范原铭[6] 杜之渝[3] 

机构地区：[1]重庆医科大学附属第一医院妇产科,重庆400016 [2]山东省胸科医院呼吸内科,济南250000 [3]重庆医科大学附属第二医院眼科,重庆400010 [4]山东淄矿集团中心医院消化内科,淄博255120 [5]山东淄矿集团中心医院普外科,淄博255120 [6]重庆医科大学附属第一医院内分泌外科,重庆400016

出　　处：《重庆医科大学学报》2011年第12期1412-1415,共4页Journal of Chongqing Medical University

摘　　要：目的:观察慢病毒载体(Lentivirus vectors,LVs)介导的增强型绿色荧光蛋白(Enhanced green fluorescence protein,eGFP)基因转染离体兔角膜上皮细胞对其生理功能的影响。方法:角膜上皮细胞的原代及传代培养并细胞鉴定。分正常细胞组(对照组)与转染细胞组。LVs介导的eGFP基因(Lenti-eGFP)以最适感染复数(Multiples of infection,MOI)=100转染角膜上皮细胞并成功传代,倒置荧光显微镜观察eGFP的表达情况。HE染色光镜观察及透射电子显微镜观察2组细胞的形态及超微结构变化;计算2组细胞在传代第2、3、4天的细胞增殖率;流式细胞技术(Flow cytometer,FCM)检测2组角膜上皮细胞凋亡率的变化。结果:eGFP于转染48 h即开始有表达,随着转染时间的延长其在角膜上皮细胞内的表达增强。细胞成功传代后,eGFP仍明显表达于角膜上皮细胞。在最适感染复数,即MOI=100时,HE染色光镜观察见转染组细胞形态规则,无异常核分裂像,与对照组角膜上皮细胞形态一致;透射电子显微镜观察转染组细胞超微结构与对照组细胞无明显差别。2组细胞分别在传代第2、3、4天的增殖率差异无统计学意义(P>0.05)。FCM检测细胞凋亡率2组间无明显差异(P>0.05)。结论:Lenti-eGFP可以有效转染离体兔角膜上皮细胞,但对上皮的影响需要进一步探讨。Objective：To observe the influence on cell physiological function after lentivirus vectors mediated enhanced green fluorescence protein（eGFP） gene transfection into rabbit corneal epithelial cells in vitro.Methods：The primary and passage rabbit corneal epithelial cells were cultured and the cells were transfected by lentivirus vectors mediated eGFP gene（lenti-eGFP） with the optimum multiplicity of Infection（MOI）=100.After successful transfection,the expression of eGFP was examined through the inverted fluorescence microscope.The cellular morphology and ultra microstructures of the epithelial cells were observed by HE stain and transmission electron microscope.Furthermore,we calculated the proliferation rate of the normal（control） and transfected cells on 2,3 d and 4 d since the culture.The apoptosis rate of both transfection cells and control cells was counted by Flow cytometer（FCM）.Results：The eGFP began to express in the cells and was observed by the inverted fluorescence microscope 48 h after transfection and was still seen after the cells were subcultured.At MOI=100,the cellular morphology and ultra microstructure of infected cells were not changed significantly compared with the normal epithelial cells.The cell proliferation rate for the two groups on 2,3 d and 4 d since the culture was 1.07±0.14 vs 1.04±0.19,0.52±0.06 vs 0.53±0.05 and 0.23±0.03 vs 0.21±0.04 respectively.There was no significant difference in the cell proliferation rate between infected cells and control cells（P0.05）.The outcome of apoptosis rate（0.645±0.040 vs 0.650±0.030） suggested no significant difference in both normal and infected cells（P0.05）.Conclusion：Lenti-eGFP could transfect the rabbit epithelial cells ex vivo efficiently and the influence on the cells needs further discussion.

关 键 词：慢病毒载体 基因转染 增强型绿色荧光蛋白 角膜上皮 细胞实验 

分 类 号：Q789[生物学—分子生物学] R322.9[医药卫生—人体解剖和组织胚胎学]