【摘要】 目的体外构建编码人类表皮生长因子受体(EGFR)的短发卡状RNA(shRNA)的质粒表达载体,观察其对结肠癌LoVo细胞EGFR的特异性抑制作用以及对细胞凋亡的影响。方法体外合成EGFR的DNA模板引物和Pgenesil-1质粒构建编码shRNA的表达载体。应用脂质体Lipofectamine2000转染人结肠癌LoVo细胞,转染成功后以G418筛选4周,实时荧光定量RT-PCR(realtime RT-PCR)和Western-blot检测EGFR的表达,流式细胞仪检测细胞凋亡。结果成功的构建了针对EGFR的质粒表达载体。质粒载体成功转染后,EGFR mRNA表达下降了(81.3±2.8)%,蛋白表达下降了(73.4±2.3)%,细胞凋亡增加了(10.1±0.4)%,和对照质粒载体比较差异有统计学意义。结论我们构建的针对EGFR的质粒表达载体可以显著抑制其在人结肠癌LoVo细胞的表达,诱导细胞凋亡,为结肠癌的基因治疗提供了新的思路。更多还原

【Abstract】 Objective To establish plasmid expressive vector coding short hairpin RNA (shRNA) of human epidermal growth factor receptor (EGFR) gene in vitro,to investigate specific inhibition of EGFR expression in human colon cancer LoVo cells and the influence of apoptosis by shRNA.Methods DNA templa-primer of EGFR was synthetized in vitro and the plasmid expressive vectors coding shRNA were established with Pgenesil-1 plasmid.The human colon cancer LoVo cells were transfected by Lipofectamine 2000 with plasmid vector,and then were selected for 4 weeks by G418.The EGFR mRNA was assessed by Real Time PCR and the protein was assessed by Western-blot,the cell apoptosis was determined via flow cytometry.Results Plasmid expressive vectors aimed directly at EGFR were successfully established.After the plasmid vectors were transfected into the LoVo cells,the EGFR mRNA expression was decreased by (81.3±2.8)%;the protein expression was decreased by (73.4±2.3) %,cell apoptosis increased by (10.1±0.4)%,respectively.There was significant difference to control plasmid vector.Conclusion The plasmid expressive vector aimed directly at EGFR in our study was capable of suppressing EGFR expression of human colon cancer LoVo cells and inducing apoptosis.The new therapeutic modalities in the treatment of human colon cancer are suggested by this study.更多还原