联合应用胸苷激酶基因和胞嘧啶脱氨酶基因对MCF-7细胞的杀伤作用

目的研究双自杀基因(胞嘧啶脱氨酶基因CD和胸苷激酶基因HSV-tk)的应用对乳腺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双自杀基因转染入PA317细胞，用其病毒上清转染乳腺癌细胞MCF-7，G418筛选出阳性细胞MCF-7／CD tk，然后加入不同浓度的5-氟胞嘧啶(5-Fc)或／和丙氧鸟苷(GCV)，MTT法观察其杀伤作用；将不同比例的MCF-7／CD tk和MCF-7细胞混合，联合应用5-Fc和GCV杀伤，观察其旁观者效应。结果单独应用5-Fc或GCV均能对MCF一7／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％时即可对半数混合细胞产生杀伤作用。结论双自杀基因的应用对乳腺癌细胞具有明显的杀伤作用．其旁观者效应明显。

Killing effects of combination of cytosine deaminase and HSV-tk genes on MCF-7 cells mediated by retroviral vector

Objective To investigate the different cell-killing and bystander effects on MCF-7 cells with double suicide genes (CD and HSV-tk) transferred by retroviral vector and to find a more efficient and low-toxicity suicide gene therapy for breast cancer.Methods CD and HSV-tk double suicide genes were transfected into PA317 cells using lipofectamine.The positive clones were picked out and cultured after G418 selected.The viral supernatant was collected and the MCF-7 cells were infected with the virus containing the double suicide genes.After G418 selection,the MCF-7/CD tk cells in culture were picked and treated with 5-Fc and/or GCV.The cytoxicity efficacy was evaluated by microculture tetrajolium test (MTT) method.Furthermore,the MCF-7/CD tk and MCF-7 cells were treated by 5-Fc and GCV at varying mixing ratio to explore the bystander effects.Results The virus containing double suicide gene was produced in PA317 cells and the cell line MCF-7/CD tk which expressed double suicide genes was acquired.The killing effect of 5-Fc or GCV on MCF-7/CD tk cells is effective and that of combination of 5-Fc and GCV is more effective than that of using 5-Fc or GCV alone.When the ratio of MCF-7/CD tk cell reached 20 %,50 % of the cells was killed.Conclusion The cell-killing efficacy of double suicide gene was better than that of single suicide gene and the bystander effect was obvious.