腺病毒介导的CD-TK融合基因联合GCV和/或5-Fc对人膀胱癌细胞的杀伤作用

目的：研究腺病毒介导的CD—TK融合基因联合前体药物对人膀胱癌细胞的杀伤作用。方法：利用含有CD—TK融合基因及绿色荧光蛋白（GFP）基因的复制缺陷腺病毒转染人膀胱癌细胞株T-24细胞,GFP表达可作为转染是否成功的间接标志,PCR扩增后琼脂糖凝胶电泳检测转染后细胞CD及TK基因序列的表达情况。用丙氧鸟苷（GCV）和／或5氟胞嘧啶（5-FC）,作为前体药物,以四甲基偶氮唑蓝（MTT）法检测其对转染后T-24细胞的杀伤作用及旁观者效应。结果：腺病毒在体外能高效转染T-24细胞,72h转染效率可接近100％。GCV和／或5-FC均能有效杀伤转染后膀胱癌细胞,给予相同浓度前体药物时,联合用药组显示出较强的肿瘤杀伤作用。0．5mg／ml GCV、0．1mg／ml 5-Fc和GCV＋5-Fc作用于转染后T-24细胞72h细胞生存率分别为23．30％、20．63％、10．10％。旁观者效应杀伤实验表明5-Fc组较GCV旁观者效应明显,而联合用药组显示出更强的旁观者效应。利用Hoechst-PI双染色法检测经前体药物作用后转染细胞,各组均可见凋亡细胞,但联合用药组凋亡细胞较单一用药组多。结论：CD—TK融合基因联合应用GCV和／或5-Fc能有效杀伤膀胱肿瘤细胞,并存在较强的旁观者效应,其杀伤机制可能与凋亡相关,为适用于人膀胱癌的治疗提供一条新思路。

Lethal effects of adenovirus-mediated CD-TK gene on human bladder tumor cell

Objective: To study the lethal effects of double suicide gene CD-TK mediated by replication defective adenovirus vector on human bladder tumor cell in vitro. Methods: To transfect the adenovirus expression vector containing CD-TK gene and green fluorescent protein (GFP) gene into the human bladder tumor cell line T-24 cells. The GFP expression was the indirect sign for successful transfection, and the PCR assay was used to make sure if the CD-TK gene was well introduced into the T-24 cells. We added the ganciclovir (GCV) and/or 5-fluorocytosine (5-Fc) into the T-24 cells that containing the CD-TK gene, and then observed the lethal effects and "bystander effect" on target cells, which were determined by MTT assay. Results: After using prodrugs, the survival rate of the infected human bladder tumor cells markedly decreased. The survival rate were 23.30%,20.63%, and 10.10% 72h after adding 0.5mg/ml GCV, 0.1mg/ml 5-Fc and GCV+5-Fc respectively. The "bystander effect" was obvious. Double prodrugs showed the more effective lethal effects and "bystander effect" than the single prodrug. Double prodrugs group showed more apoptosis cells than the single prodrug group according to Hoechst-PI colorant analysis. Conclusions: CD-TK gene and GCV/5-Fc are lethal for human bladder tumor cells, while the bystander effect was showed. Apoptosis is perhaps one of the mechanisms of the killing effect. It is possible to use the double suicide gene to heal the bladder tumor.