摘要
目的构建针对人胃泌素基因的shRNA干扰表达载体,观察胃泌素基因表达抑制对人胃癌细胞系BGC823细胞增殖和迁移能力的影响。方法根据人胃泌素序列设计并构建3个靶向人胃泌素基因的shRNA真核表达载体,以脂质体转染的方法将胃泌素干扰质粒转染至人胃癌细胞系BGC823,经G418筛选出稳定转染的阳性细胞。通过RT-PCR技术筛选出表达抑制最强的稳定转染胃癌细胞系。应用流式细胞仪技术检测其细胞周期变化情况。采用Trans-well小室法检测细胞体外迁移能力。结果经测序证明胃泌素shRNA基因已成功插入Psilencer3.1质粒中,构建的干扰质粒能够显著抑制胃泌素的表达。胃泌素基因的表达下调将胃癌细胞阻滞在G1期,且穿过Trans-well小室膜的细胞数显著下降。结论干扰胃泌素的表达可显著抑制胃癌细胞的增殖和迁移能力。
Objective To construct shRNA expression vector target human gastrin gene and to investigate the effect of short hairpin RNA （shRNA） targeting on the cell cycle of gastric cancer BGC - 823 cells. Methods shRNA expressing vectors targeting to human gastrin were designed and successfully constructed. Transfectin of recombinant in gastric cancer cells BGC -823, and the stable transfectant were selected by G418. The mRNA level of gastrin expression were analyzed by RT - PCR. Migration capability of stably transfected cells in vitro was evaluated by using Transwell chamber model. FCM was performed to detect the cell cycle. Results The recombinant plasmid vector was identified by sequence analysis. RT - PCR showed that gastrin - shRNA could significantly down - regulate gastrin mRNA expression in BGC -823 cells. BGC823 cells were arrested in G1 phase and the trans -membrane cell number decreased significantly. Conclusion Knock down of gastrin gene could suppress gastric cancer proliferation and diversion by decreasing its migration capability.
出处
《青海医学院学报》
CAS
2010年第1期1-5,23,共6页
Journal of Qinghai Medical College
