草酸铂、5-氟尿嘧啶联合多药耐药基因反义RNA对耐药直肠癌细胞杀伤作用的研究

The lethal effect of combined MDR1 anti-sense RNA with oxaliplatin and 5-FU on drug-resistant rectal carcinoma cells

目的应用基因克隆技术,将多药耐药基因(multidrugresistancegene,MDR1)反义RNA转染入对氟尿嘧啶(5-FU)耐药直肠癌细胞(8348R)中,封闭正义MDR1的转录和表达,联合应用草酸铂及5-FU共同作用于8348R,观察其联合杀伤作用。方法构建含MDR1反义RNA的真核表达质粒PC-MDR1;以绿色荧光蛋白(greenfluorescenceprotein,GFP)基因作为报告基因,观察在草酸铂作用下GFP基因对8348R细胞的转染效率,用克隆形成试验观察草酸铂对PC-MDR1质粒转染8348R细胞的影响;用MTT试验检测草酸铂联合5-FU及MDR1反义RNA对8348R细胞的杀伤效率。结果转染PC-MDR1质粒后,8348R细胞在5-FU作用下较转染前活性明显下降,转染后细胞出现明显的S期和G2/M期阻滞,细胞凋亡比例显著升高;草酸铂将PC-MDR1质粒对直肠癌细胞的转染效率提高18倍,IC50剂量草酸铂、5-FU联合MDR1反义RNA可大大提高对8348R细胞的杀伤效率,总体杀伤效率可达到75%。结论应用草酸铂、5-FU联合MDR1反义RNA对直肠癌耐药细胞的协同杀伤作用,可望成为治疗对5-FU耐药的直肠癌的有效方法。

Objective To observe the lethal effect of multidrug resistance gene （ MDR1 ） antisense RNA combined with oxaliplatin and 5-FU on drug-resistant rectal carcinoma cells. Methods PC-MDR1 plasmid including MDR1 was constructed with gene cloning techniques. The drug-resistant cancer cells （8348R） were transferred with the plasmids, and the positive neoplasm cells were selected with G418. Green fluorescent protein（GFP） gene was used as a reporting gene to monitor the gene transfer efficiency under the influence of oxaliplatin and 5-FU. The cytotoxicity and therapeutic effects of MDR1 anti-sense RNA combined with oxaliplatin and 5-FU were evaluated by colony-forming rate and MTT assay. Results A significant decrease of biological activity was observed in 8348R cells transferred with PC-MDR1, cell cycles were blocked in S phase, or in G2/M phase, and apoptosis rate of the cells increased. With treatment of oxaliplatin, the plasmid transfer efficiency in the drug-resistant cancer cells was improved about 18 times. Using an ICso dose of oxaliplatin and 5-FU combined with （ MDR1 ） anti-sense RNA, 75 percent of 8348R cells were killed, which was significant higher than that of the control cells. Conclusions Combined MDR1 antisense RNA with oxaliplatin and 5-FU has a synergistic effect of killing drug-resistant cancer cells and may be a promising method for treating drug-resistant rectal carcinoma.