慢病毒介导的单纯疱疹病毒胸苷激酶基因／丙氧鸟苷体系抑制人晶状体上皮细胞的研究  

The effect of ientivirus-mediated suicide gene therapy on lens epithelial cells

作　　者：杨晋[1] 卢奕[1] 郭礼和[2] 刘天津[2] 莫晓芬[1] 

机构地区：[1]复旦大学附属眼耳鼻喉科医院眼科,200031 [2]中国科学院上海细胞生物学研究所

出　　处：《中华眼科杂志》2007年第9期810-816,共7页Chinese Journal of Ophthalmology

基　　金：上海市自然科学基金资助项目（044119659）

摘　　要：目的探讨慢病毒介导的单纯疱疹病毒胸苷激酶基因／丙氧鸟苷体系（HSV—tk／GCV）对人晶状体上皮细胞（LECs）系的杀伤效应的机制。方法HSV—tk基因和增强型绿色荧光蛋白（EGFP）报告基因共表达的慢病毒感染细胞为试验组，仅表达EGFP的慢病毒感染细胞和正常细胞为对照组。流式细胞仪检测病毒的转染效率，荧光显微镜观察及基因组PCR和RT-PCR检测基因在LECs内表达，DNA ladder和电镜观察该系统对LECs的杀伤作用，检测HSV—tk／GCV系统的浓度时间依赖性和旁观者效应。结果HSV—tk整合入LECs并高效表达。10～25μg／ml的GCV能明显诱导转染HSV—tk的细胞凋亡或坏死，且随GCV浓度升高和作用时间延长效应增强，且存在明显的旁观者效应，而对照组细胞无显著改变。当GCV浓度〉25μg／ml，对照组细胞的增殖也被显著抑制。结论GCV可高效杀伤表达HSV-tk的LECs，且旁观者效应显著增强了HSV—tk／GCV系统的杀伤效果。HSV—tk和EGFP共表达的慢病毒载体能高效稳定转染LECs。Objective To investigate the cytotoxicity of lentivirus-mediated herpes simplex virus thymidine kinase/ganciclovir （HSV-TK/GCV） suicide gene therapy on human lens epithelial cell line and analyze the mechanism of cell death. Methods a lentiviral containing the Lenti-HSVtk-EGFP as therapeutic vector and Lenti-EGFP as the control were used in the study, Transfection efficiency in vitro was assessed by fluorescence-activated cell sorting, Expression of HSV-tk in lens epithelial cells （LECs） mediated by lentivirus was examined by fluorescence microscope, genomic PCR and reverse transcription PCR. The cytotoxicity of HSV-TK/GCV suicide-gene system was assessed using DNA ladder and electron microscope, The time dependent transfection efficiency and bystand effect induced by the HSV-TK/GCV in LECs were evaluated, Result the transduction efficiency was higher than 95%, When concentration of GCV was 15-25 μg/ml, apoptosis or necrosis was induced by Lenti-HSVtk-EGFP in HLE, The cytotoxicity was enhanced with increased time of transfection and concentration of GCV, Non transfected cells were also effectively killed by mixing the cell with GCV transfected cells （ Bystander effect ）, Conclusion GCV can effectively kill the LECs with the expressing of HSV-tk, Bicistronie lentiviral vectors can effieiendy integrate multiple genes into LECs, therefore, it is a reliable vector for gene therapy; lentivirus mediated HSV-tk/GCV suicide gene therapy may provide an effective approach for the treatment of posterior capsule opacification.

关 键 词：基因 转基因 自杀 慢病毒属 发光蛋白质类 单纯疱疹病毒属 胸苷激酶 晶体 上皮细胞 

分 类 号：R686[医药卫生—骨科学]