论文部分内容阅读
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。
Objective To construct a plasmid expression vector encoding short hairpin RNA (shRNA) of human epidermal growth factor receptor (EGFR) in vitro and observe the specific inhibitory effect on EGFR of colon cancer LoVo cells and its effect on apoptosis. Methods EGFR DNA template primers and Pgenesil-1 plasmid were constructed in vitro to construct shRNA expression vector. Lipofectamine 2000 was used to transfect human colon cancer LoVo cells. After transfection, the cells were screened by G418 for 4 weeks after transfection. Real-time RT-PCR and Western-blot were used to detect the expression of EGFR. Flow cytometry Apoptosis. Results A plasmid expression vector targeting EGFR was successfully constructed. The expression of EGFR mRNA decreased by (81.3 ± 2.8)%, the expression of EGFR decreased by (73.4 ± 2.3)% and the apoptosis increased by (10.1 ± 0.4)% after plasmid vector was transfected successfully, which was statistically different from the control plasmid vector Significance of learning. Conclusion Our constructed plasmid expression vector targeting EGFR can significantly inhibit its expression in human colon cancer LoVo cells and induce apoptosis, providing a new idea for the gene therapy of colon cancer.