腺病毒介导TK/GCV基因系统联合肿瘤坏死因子对膀胱癌细胞的体外杀伤作用  

Effect of adenovirus-mediated TK/GCV gene therapy in combination with TNF-α against murine bladder cancer cells in vitro

作　　者：石向华[1] 谭万龙[1] 朱文辉[1] 梁中锟[1] 杜跃军[1] 

机构地区：[1]南方医科大学南方医院泌尿外科,广东广州510515

出　　处：《南方医科大学学报》2008年第5期750-753,757,共5页Journal of Southern Medical University

基　　金：广东省自然科学基金(020059)~~

摘　　要：目的研究腺病毒介导TK/GCV系统联合肿瘤坏死因子(TNF-α)对膀胱癌细胞的体外杀伤作用。方法采用含有TK基因、绿色荧光蛋白(GFP)基因的复制缺陷腺病毒(adv+)转染MB49细胞,观测转染率,RT-PCR检测转染细胞TK基因产物。以MB49细胞为对照,测定不同浓度TNF-α作用下MB49细胞、不同浓度GCV作用下MB49/TK细胞的存活率,采用GCV联合低浓度TNF-α对MB49/TK细胞进行体外杀伤研究;流式细胞仪检测TK/GCV、TNF-α、TNF-α+TK/GCV对MB49细胞作用8h后细胞凋亡情况。结果随着浓度的增高GCV对MB49/TK细胞、TNF-α对MB49细胞的生长抑制率逐渐增高。GCV联合TNF-α对细胞杀伤率均较同浓度下单纯GCV及单纯TNF-α作用时的细胞杀伤率有明显增强,且随TNF-α浓度增高联合治疗组杀伤效率增强:单纯50μg/mlGCV组、5μg/mlTNF-α+50μg/mlGCV组、20μg/mlTNF-α+50μg/mlGCV组杀伤率分别(24.39±1.10)%、(40.05±0.97)%、(65.47±0.67)%。流式细胞仪检测细胞周期示TK/GCV、TNF-α、TK/GCV+TNF-α组作用细胞8h均可见典型sub-G1期细胞凋亡峰,联合作用组凋亡率最高。结论TNF-α能明显增强TK/GCV自杀基因系统对膀胱癌的杀伤作用,两者联合能够有效促进膀胱癌细胞的凋亡。ObjeCtive To investigate the cell-killing effect of adenovirus-mediated TK-ganciclovir （GCV） gene therapy in combination with tumor necrosis factor-α （TNF-α） against murine bladder carcinoma cells in vitro. Methods Murine bladder carcinoma MB49 cells were transfected with the adenoviral vector containing TK gene and green fluorescent protein （GFP） gene. The transfection efficiency was observed and the TK gene expression in the transfected cells was detected by RT-PCR. The survival rate of MB49 cells in response to TNF-α treatment and that of the TK gene-transfected cells after treatment with GCV and GCV＋TNF-α were determined by MTT assay. The apoptosis of the cells after the treatments was analyzed by flow cytometry. Results In cells transfected with TK gene, the cell inhibition rate increased gradually with the increment of GCV and TNF-α concentration. GCV in combination with TNF-α resulted in significantly increased killing efficiency of the cells as compared with GCV or TNF-α treatment alone, and the effect of the combined treatment was enhanced as the TNF-α concentration increased. GCV treatment （50 μg/ml） alone produced a cell killing rate of （24.39±1.10）%, and when combined with 5μg/ml TNF-α, the rate was increased to （40.05±0.97）%, and further to （65.47±0.67）% when TNF-α concentration increased to 20 μg/ml. Flow cytometry revealed obvious apoptosis of the cells 8 h after treatments with TK/GCV, TNF-α, or TK/GCV＋TNF-α, and the combined treatment resulted in the highest cell apoptotic rate. Conclusion TK/GCV in combination with TNF-α can enhance the effect of suicide gene therapy against murine bladder carcinoma cells and effectively induce apoptosis of the cells.

关 键 词：基因治疗 TK基因 TNF-α 膀胱癌 细胞凋亡 

分 类 号：R737.14[医药卫生—肿瘤]