腺相关病毒介导HSV-tk/GCV系统对晶状体上皮细胞的旁观者效应及机制  

Bystander effect of HSV-tk/GCV system mediated by adeno-associated virus on lens epithelial cells in vitro and its mechanism

作　　者：丁芝祥[1] 彭燕一[1] 邱梅园[1] 张玉明[1] 曾新生[1] 

机构地区：[1]桂林医学院附属医院眼科,广西壮族自治区桂林市541001

出　　处：《眼科新进展》2012年第8期708-711,共4页Recent Advances in Ophthalmology

基　　金：广西自然科学基金资助(编号:0899004)~~

摘　　要：目的研究2型重组腺相关病毒(recombinant adeno-associated virus2,rAAV2)载体介导的单纯疱疹病毒胸苷激酶基因/丙氧鸟苷(HSV-tk/GCV)系统对体外培养兔晶状体上皮细胞(N/N1003A)的旁观者效应,并初步探讨其机制。方法 rAAV2载体介导增强型绿色荧光蛋白基因(enhanced green fluorescent,EGFP)转染体外培养N/N1003A细胞,倒置荧光显微镜观察细胞中EGFP的表达,流式细胞仪检测病毒的转染效率。重组病毒rAAV2/HSV-tk转染体外培养的N/N1003A细胞(N/N1003A-tk),以不同比例与没有转染的细胞混合,MTT法观察旁观者效应;MTT法检测N/N1003A-tk/GCV处理的条件培养液(CM)对N/N1003A细胞的作用。应用相差显微镜、透射电镜观察条件培养液对N/N1003A细胞形态学和超微结构的影响。结果 rAAV2载体能介导EGFP基因稳定转染N/N1003A细胞,转染效率最高达81.12%。当N/N1003A-tk细胞以不同比例(10%、20%、40%、80%、100%)与N/N1003A混合时,细胞抑制率分别为49.00%±0.49%、54.71%±1.41%、59.26%±1.13%、68.20%±0.76%、73.44%±1.26%,与对照组(1.68%±0.45%)比较,差异均有显著统计学意义(均为P<0.01)。N/N1003A-tk/GCV处理的条件培养液可明显杀伤N/N1003A细胞,但将其用0.22μm滤膜过滤后,对细胞的杀伤作用消失。条件培养液处理的N/N1003A细胞出现凋亡征象。结论 rAAV2介导的HSV-tk/GCV系统对兔晶状体上皮细胞有较强的旁观者效应,通过非细胞接触方式诱导旁观者细胞凋亡可能是其机制之一。Objective To study the bystander effect of recombinant adeno-associated virus 2（rAAV2）-mediated herpes simplex virus thymidine kinase/ganciclovir（HSV-tk/GCV） system on rabbit lens epithelial cells（N/N1003A） in vitro,and investigate its mechanisms.Methods After N/N1003A cells were transfected with rAAV2-enhanced green fluorescent protein（EGFP）,expression of EGFP was observed by inverted fluorescent microscope,and the transfection efficiency was detected by flow cytometry.After N/N1003A cells infected by recombinant virus rAAV2/HSV-tk（N/N1003A-tk） were mixed with N/N1003A cells in different proportion,bystander effect was observed by MTT assay.The effect of conditioned medium（CM） of N/N1003A-tk/GCV on N/N1003A cells was detected by MTT assay.The changes of morphology and ultrastructure of N/N1003A cells treated by CM were studied with phase contrast microscope and transmission electron microscope.Results rAAV2 vector encoding EGFP gene could be transfected into N/N1003A cells stably,and the highest transfection efficiency was 81.12%.When N/N1003A-tk cells were mixed with N/N1003A cells in different proportion（10%,20%,40%,80% and 100%）,the cell inhibition rates were 49.00%±0.49%,54.71%±1.41%,59.26%±1.13%,68.20%±0.76% and 73.44%±1.26%,respectively,and compared with the control group（1.68%±0.45%）,the differences were statistically significant（both P〈0.01）.CM of N/N1003A-tk/GCV could obviously kill N/N1003A cells,but when filtrated with 0.22 μm membrane,the effect disappeared.The N/N1003A cells treated by CM of N/N1003A-tk/GCV appeared apoptosis.Conclusion HSV-tk/GCV system mediated by rAAV2 has strong bystander effects on rabbit lens epithelial cells in vitro,and bystander cells’ apoptosis induced by non-contact way may be one of its mechanisms.

关 键 词：旁观者效应 单纯疱疹病毒胸苷激酶 晶状体上皮细胞 腺相关病毒 

分 类 号：R737.14[医药卫生—肿瘤]