腺病毒介导的HSV-TK基因系统联合羟基喜树碱对人膀胱癌细胞的体外杀伤作用  

Lethal effect of adenovirus-mediated HSV-TK gene in combination with hydroxycamptothecin on human bladder cancer in vitro

作　　者：黄河[1] 谭万龙[1] 朱文辉[1] 梁仲琨[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》2007年第4期461-464,共4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的HSV-TK系统联合羟基喜树碱(HCPT)对人膀胱癌细胞的体外杀伤作用。方法使用含有HSV-TK基因、绿色荧光蛋白(GFP)基因的复制缺陷腺病毒转染人膀胱癌细胞T-24细胞,表达GFP为转染成功标志,同时观察转染率,PCR检测TK表达。以正常T-24细胞为空白对照,转染成功后分别加入GCV、GCV+HCPT,采用MTT法测定单纯HCPT作用于人膀胱癌细胞72h细胞增殖抑制率、各组治疗转染后人膀胱细胞24、48、72h细胞存活率;流式细胞仪检测GCV(0.5mg/ml)+HCPT(10mg/L)作用T-24细胞4h后的凋亡情况。结果随着浓度的增高,HCPT对T-24细胞的生长抑制率逐渐增高,HCPT浓度为0.01mg/L时T-24生长抑制率为14%,50mg/L时T-24生长抑制率为63%,当HCPT浓度大于100mg/L时,抑制作用无明显增加(P=0.216)。GCV组、GCV+HCPT各组对转染后细胞有明显杀伤作用(P=0.00),GCV+HCPT组随HCPT浓度增高和作用时间的延长,杀伤效率明显增高。0.5mg/mlGCV单独作用于转染后的膀胱癌细胞72h后,细胞存活率为34.6%,GCV(0.5mg/ml)+HCPT(10mg/L)组的细胞存活率仅为8.07%(P=0.00)。GCV+10mg/mlHCPT作用后4h,T-24细胞出现M1期前典型的细胞凋亡峰,凋亡率达52.93%。结论HSV-TK/GCV系统联合HCPT治疗能增强自杀基因系统对人膀胱癌的杀伤作用,能够良好地诱导人膀胱癌细胞的凋亡。Objective To evaluate the lethal effect of adenovirus-mediated HSV-TK-ganciclovir （GCV） gene therapy in combination with hydroxycamptothecin （HCPT） on hunman bladder carcinoma cell line T-24 cells. Methods Hunman bladder carcinoma cell line T-24 was transfected with adenovirus expression vector containing TK gene and green fluorescent protein （GFP） gene, and the transfection efficiency was observed and TK expression detected by PCR. After successful cell transfection indicated by GFP expression, GCV and hydroxycamptothecin are respectively added into the cell culture with normal T-24 cells serving as the blank control group. The growth inhibition rate of hunman bladder carcinoma cells in response to HCPT treatment for 72 h and the cell survival rate of 24 h, 48 h and 72 h after transfection with different protocols were observed by MTT assay. The apoptosis of the cells treated with GCV （0.5 mg/ml）＋HCPT （10 mg/L） for 4 h was observed by flow cytometry. Results The cell inhibition rate increased gradually with increment of HCPT concentration, from 14% at HCPT concentration of 0.01 mg/L to 60% at 50 mg/L, but for a concentration above 100 mg/L, the inhibition rate did not exhibit further increase （P=-0.216）. GCV alone and GCV in combination with HCPT both resulted in significantly decreased survival rate of human bladder carcinoma cells （P=0.00）, and the killing efficiency of the cells by GCV＋HCPT protocol increased obviously with increment of HCPT concentration and prolongation of the action time. The cells treated with 0.5 mg/ml GCV alone for 72 h retained a cell survival rate of 34.6%, which was lowered to only 8.07% with combined treatment with GCV （0.5mg/ml） and HCPT （10 mg/L）. Typical apoptotic peak before M1 phase of the cells appeared 4 h after treatment with GCV＋10 mg/ml HCPT, which resulted in a apoptosis rate of 52.93%. Conclusion HSV-TK/GCV in combination with HCPT can enhance the lethal effect of suicide gene therapy against human bladder carcinoma cells and eff

关 键 词：基因治疗 TK 膀胱癌 细胞凋亡 

分 类 号：R737.14[医药卫生—肿瘤]