摘要
目的构建针对bcl-2基因的短发卡RNA(shRNA)干扰真核质粒表达载体,转染到bcl-2基因高表达的胃癌细胞株SGC-7901中,并筛选出稳定低表达bcl-2基因的细胞株。方法针对bcl-2基因的mRNA序列设计、合成4对寡核苷酸序列,插入质粒载体pGPH1/GFP/Neo中,经脂质体介导转染SGC-7901细胞。RT-PCR检测bcl-2基因在mRNA水平的变化,MTT法检测bcl-2基因沉默后胃癌SGC-7901细胞的增殖情况。基因沉默效果最好的一组,经G418筛选以得到稳定表达株。结果与对照组相比,转染成功后的细胞bcl-2基因在mRNA水平均显著下降,细胞增殖速度明显降低(t=2.41,P<0.05);并筛选出稳定表达shRNA2质粒的阳性克隆。结论本研究成功利用针对bcl-2基因的shRNA质粒载体筛选出稳定低表达bcl-2基因的SGC-7901细胞,为胃癌的基因治疗研究奠定了基础。
Objective To construct a short, bcl 2 gene-targeting hairpin RNA （shRNA） plasmid vector, which was transfected to high-bcl-2-gene-expressed gastric cancer line SGC-7901, a ceil strain with stable and low bcl 2 gene expression was then screened. Methods Four pairs of oligonucleotide specific for bcl 2 gene were designed, synthesized, and inserted into pGPH1/GFP/Neo plasmids. The vectors were transfected into SGC-7901 cells with lipofectamine. RTPCR was used to detect bel- 2 gene expression and MTT technique employed to detect the proliferation of tumor cells after bcl-2 gene was silenced. Ceils with the strongest inhibition were selected with G418 and the stable cell clones obtained. Results As compared with the control, the mRNA expression of bcl-2 in the transfected cells was significantly suppressed, the rate of cell proliferation was significantly reduced （t=2.41 ,P〈0.05）. Conclusion SGC-7901 cell strains, which express stable and lower level of bci-2 mRNA, were successfully obtained by using bcl 2-targeting shRNA plasmid vector. The present study provided basis for research on gene therapy of gastric cancer.
出处
《青岛大学医学院学报》
CAS
2010年第2期95-97,100,共4页
Acta Academiae Medicinae Qingdao Universitatis
