联合应用胸苷激酶基因和胞嘧啶脱氨酶基因对胆囊癌细胞的杀伤作用

目的研究双自杀基因的应用对胆囊癌细胞的杀伤作用和旁观者效应。方法脂质体法将CD和HSV-tk双自杀基因转染人PA317细胞，用其病毒上清转染胆囊癌细胞GBC-SD，G418筛选出阳性细胞(GBC-SD/CD tk，应用不同浓度的5-FC或(和)GCV，MTT法观察其杀伤作用；将不同比例的GBC-SD/CD tk和GBC-SD细胞混合，联合应用5FC和GCV杀伤，观察其旁观者效应。结果单独应用5FC或GCV均能对转染阳性的GBC-SD／CD tk细胞产生明显的杀伤效应，联合应用可以在较小的剂量下产生更大的杀伤作用；当阳性细胞比例达到20％时即可对半数细胞产生杀伤作用。结论双自杀基因的应用对胆囊癌细胞具有明显的杀伤作用，其旁观者效应明显。

Killing effects of cytosine deaminase gene in combination with HSV-tk gene on gallbladder cancer cells

Objective To investigate the different cell-killing and bystander effects on GBC-SD cells with double suicide genes (cytosine deaminase and HSV-tk) transferred by retroviral vector and to find a more efficient and low-toxicity suicide gene therapy for gallbladder cancer. Methods Cytosine deaminase (CD) and HSV-tk double suicide genes were transfected into PA317 cells using lipofectamine. The positive clones were picked out and cultured after G418 was selected. The viral supernatant was collected and the GBC-SD cells were infected with the virus containing the double suicide genes. After G418 selection, the GBC-SD/CD+tk cells were collected and treated with 5-FC and/or GCV. The cytotoxicity efficacy was evaluated by MTT. Furthermore, the GBC-SD/CD+tk and GBC-SD cells were treated by 5-FC and GCV at different mixing ratios to explore their bystander effects. Results The virus containing double suicide gene was produced in PA317 cells and the cell line GBC-SD/CD+tk that expressed double suicide genes was acquired. The killing effect of 5-FC or GCV on GBS-SD/CD+tk cells was effective. The killing effect was more effective by using 5-FC in combination with GCV than by using 5-FC or GCV alone. When the ratio of GBC-SD/CD+tk cells reached 20%, 50% of the cells were killed. Conclusions The cell-killing efficacy of double suicide genes is higher than that of single suicide gene. Meanwhile, its bystander effect is obvious.