X线联合胞嘧啶脱氨酶基因对结肠直肠肿瘤细胞的杀伤作用

X-ray combined with cytosine deaminase suicide gene therapy enhances killing of colorectal carcinoma cells in vitro

目的 提高基因疗法对结肠直肠肿瘤的疗效。 方法 以绿色荧光蛋白 (GFP)基因作为报告基因 ,观察在X线作用下GFP基因对结肠直肠肿瘤细胞的转染效率及表达时间 ;用克隆形成试验观察X线对胞嘧啶脱氨酶 (CD)基因转染的影响 ;同时用细胞存活率测定试验检测X线联合CD和 5 氟胞嘧啶对结肠直肠肿瘤细胞的杀伤作用。 结果 X线提高超螺旋质粒对结肠直肠肿瘤细胞的转染效率 2～ 4倍 ,可提高线性质粒转染效率 3 0倍。肿瘤细胞的存活率 :转染CD基因组LoVo细胞为 85 % ,83 4 8细胞为 88% ;剂量X线联合空载体组为 10 % ;LD90 剂量X线联合CD基因组 ,LoVo细胞为 9% ,83 4 8细胞为 11% ,加上X线作用 ,细胞总体存活率仅为 1%。 结论 X线联合自杀基因可有效地杀伤结肠直肠肿瘤细胞 。

Objective To study the effect of X ray on gene transfer and the antitumoral effect of X ray combined with suicide gene therapy on colorectal carcinoma cells. Methods Green fluorescent protein(GFP) was seen under fluorescent microscope.GFP gene was used for reporting gene to learn gene transfer efficiency and gene expressing time under the influence of radiation. G418 was used to select cytosine deaminase (CD) positive neoplasm cells and CD gene transfer efficiency was tested by cloning efficiency. Antitumoral effect of X ray combined with CD and 5 FC on colorectal carcinoma cells was tested by MTT. Results 4 Gy radiation could improve supercoiled plasmid DNA transfer efficiency for about 2 4 times and 30 times for linearized plasmid DNA.The mean durations of GFP gene expression treated with 4 Gy radiation were 14 d for supercoiled plasmid and 21 and for linearized plasmid,while in control group,the time was 12 d. Middle dose radiation combined with CD and 5 FC could kill 99 percent of colorectal carcinoma cells, while in the control group,5 FC only killed 15 percent of colorectal carcinoma cells which were transduced with CD gene. Conclusions X Ray combined with suicide gene therapy may be used as a promising method for treating colorectal neoplasm.