人FoxA1 shRNA载体构建与检测  

Construction and evaluation of human FoxA1 shRNA vector

作　　者：文俏程[1] 张蔚林[1] 陈志康[1] 曹小霞[2] 陈子华[1] 谭思创[3] 肖献忠[2] 谭斯品[4] 

机构地区：[1]中南大学湘雅医院普外科,湖南长沙410008 [2]中南大学湘雅医学院基础医学院病理生理学系,湖南长沙410008 [3]中南大学湘雅二医院心胸外科,湖南长沙410008 [4]中南大学细胞与分子生物学实验教学中心,湖南长沙410008

出　　处：《中国现代医学杂志》2014年第4期1-6,共6页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30800550);湖南省自然科学基金(No:10JJ4016)

摘　　要：目的构建有效的针对人FoxA1基因的shRNA干扰载体,并验证其干扰效果。方法设计合成3对针对人FoxA1基因的和1对无同源性的shRNA片段,将合成的shRNA片段插入到干扰载体pRNAT-U6.1/Neo中,经过酶切和测序验证,成功构建人FoxA1基因的shRNA和空白对照载体。将各干扰载体和空白载体转染人正常胃黏膜上皮细胞(GES-1)和胃癌细胞(SGC-7901),Realtime RT-PCR和Western-blotting检测干扰载体的干扰效率。结果①酶切和测序验证均表明各shRNA载体构建成功。将空载体及各干扰载体分别转染人正常胃黏膜细胞(GES-1)和胃癌细胞(SGC-7901)发现,和空白对照组相比,3组干扰载体能够从mRNA和蛋白表达水平明显抑制FoxA1的表达,但是3组质粒的抑制效率有一定的差异,以3号质粒对Foxa1的干扰效率最强。结论成功构建了三个有效的针对人FoxA1基因的shRNA干扰载体,三组质粒都能有效地抑制FoxA1在GES-1细胞和SGC-7901细胞中的表达。【Objective】To construct effective shRNA（short hairpin） recombinant plasmids targeting human FoxA1 gene, and evaluatethe interference efficiency of the Foxa1-shRNA plasmids. 【Methods】Three pairs of shRNA sequences targeting human FoxA1 gene and one pair of control shRNA sequence were designed, synthesized, and then inserted into the pRNAT-U6.1/Neo vector. The shRNA recombinant vectors were evaluated by enzyme digestion and sequencing. Foxa1 and control shRNA plasmids were transfected into human normal gastric epithelial cells（GES-1） and human gastric cancer cells（SGC-7901） cells, real-time RT-PCR and Western-Blotting analysis were performed to evaluate its interfering efficiency. 【Results】Restriction enzyme digestion and sequencing showed that the shRNA vectors were constructed successfully. Foxa1 mRNA and protein expression were reduced to a significant degree after transfection with the three Foxa1-shRNA plasmids, and the Foxa1-sh3 plasmid can inhibit the Foxa1 expression most efficiently. 【Conclusion】The shRNA interference vectors targeting human FoxA1 gene were successfully constructed, and the three shRNA plasmids can significantly inhibit the Foxa1 expression in GES-1 and SGC-7901 cells with different inhibition efficiency.

关 键 词：FoxA1 shRNA载体 干扰效率 

分 类 号：R735.2[医药卫生—肿瘤]