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目的自杀基因具有能将无细胞毒性的前体药物转化为有细胞毒性作用的药物而使细胞死亡,通过观察大肠杆菌胞嘧啶脱氨酶/氟胞嘧啶(CD/5-FC)自杀基因系统对食管癌EC109细胞杀伤效应及与放射联合应用对食管癌肿瘤细胞协同杀伤效应。方法采用PCR方法从大肠杆菌基因组DNA中扩增出CD基因;构建重组真核载体pcDNA3.1-CD;用脂质体转染法转染EC109细胞;观察CD/5-FC体系对EC109细胞的杀伤效应和旁观者效应及对放射的增敏效应。结果RT-PCR分析结果表明CD基因已转入EC109细胞并表达。离体实验证实5-FC对转CD基因后的食管癌细胞株有明显的细胞毒作用,含5%的转基因食管癌细胞加入5-FC后的细胞存活比率为41.8%±14.2%,低于对照组的94.6%±4.3%结果(t=3.14,P<0.05);加入10%的转基因细胞的存活比率为37.8%±4.4%,低于对照组的95.6%±5.4%结果(t=9.75,P<0.01)。CD/5-FC体系对肿瘤细胞放射增敏效果明显,加前药5-FC及放射组细胞存活曲线低于单纯加前药5-FC对照组或单纯放射对照组,重复测量数据方差分析结果显示F=11.50,P<0.01;治疗组分别与5-FC对照组及单纯放射对照组相比F值分别为4.11、10.53,P值分别<0.05、0.01。结论CD/5-FC体系的旁观者效应和放射增敏效果明显。
Purpose Suicide gene has the ability to convert non-cytotoxic prodrug into cytotoxic drug and cause cell death. By observing the CD / 5-FC suicide gene system of E. coli, The killing effect of esophageal EC109 cells and its synergistic killing effect on esophageal cancer cells in combination with radiation. Methods The CD gene was amplified by PCR from E.coli genomic DNA. The recombinant eukaryotic vector pcDNA3.1-CD was constructed and transfected into EC109 cells by lipofectamine. The effect of CD / 5-FC on EC109 cells Killing effect and bystander effect and sensitizing effect to radiation. Results RT-PCR results showed that the CD gene was transferred into EC109 cells and expressed. In vitro experiments showed that 5-FC had obvious cytotoxic effect on esophageal cancer cell lines after transfection with CD gene. The cell survival rate of 5% transgenic esophageal cancer cells with addition of 5-FC was 41.8% ± 14.2% The survival rate of the control group was 94.6% ± 4.3% (t = 3.14, P <0.05). The survival rate of the 10% transgenic cells was 37.8% ± 4.4%, which was lower than that of the control group (95.6% ± 5.4% , P <0.01). CD / 5-FC system had obvious radiosensitizing effect on tumor cells. The survival curve of 5-FC plus radiotherapy group was lower than that of 5-FC control group or radiotherapy alone group. The results of repeated measures ANOVA F = 11.50, P <0.01. The F values in the treatment group compared with the 5-FC control group and the radiotherapy alone group were 4.11 and 10.53, respectively, with P values of <0.05 and 0.01 respectively. Conclusion The bystander effect and radiosensitization effect of CD / 5-FC system are obvious.