幽门螺杆菌ureB基因转染胃上皮细胞及其对细胞的作用

Effect of recombinant plasmid of Helicobacter pylori ureB gene on gastric epithelial cell

研究幽门螺杆菌 (Helicobacterpylori,Hp)ureB基因重组子转染胃上皮细胞后对胃上皮细胞的作用。用PCR方法从Hp标准株NCTC116 37中获取ureB全长基因 ,将其开放读码框架定向克隆入真核表达载体pcDNA3 1;获得的重组子转染SGC 790 1细胞 ,筛选耐潮霉素的细胞克隆 ,用RT PCR方法检测细胞内ureB基因在转录水平的表达 ;分别用荧光染色技术、MTT、流式细胞术检测UreB对细胞表型、增殖、凋亡及细胞周期的影响。UreB阳性表达的细胞 (SureB)胞膜出芽、细胞皱缩 ;用MTT法检测细胞增殖 ,结果表明 ,SureB细胞与SpcDNA3 1细胞比较 (pcDNA3 1转染的细胞 ) ,生长增殖无显著性差异 (P >0 0 5 ) ,流式细胞术检测细胞凋亡结果显示 ,SureB的凋亡率显著高于SpcDNA3 1(P值为 0 0 0 7) ;细胞周期分析显示 ,SureB细胞有S期比率增高、G2 M、G0 G1 期比率下降的趋势。

To investigate the effect of recombinant plasmid of Helicobacter pylori ureB gene on gastric epithelial cell. The full length sequence of ureB gene from NCTC11637 was amplified by PCR. The recombinant plasmid was constructed by cloning the open reading frame (ORF) of ureB into the eukaryotic expression vector pcDNA3.1, and was transfected SGC-7901 cells, then the clones resisting Hygromacine were screened. mRNA expression of ureB of transfected cells was detected by RT-PCR.The effect of recombinant plasmid of Hp ureB gene on cell phenotype was observed by fluorescence strain, on proliferation by MTT, on apoptosis and cell cycles by flow cytometry, respectively. The positive clones of ureB(SureB) appeared cell membrane budding and cell shrinkage. MTT assay showed there was no statistic significance between the SureB and SpcDNA3.1 which were transfected only by pcDNA3.1 (P>0.05), suggesting that the growth of SureB were not inhibited. The apoptosis rate of SureB was higher than that of SpcDNA3.1(P=0.007). Analysis for cell cycle showed that in SureB cells the proportion of S phase increased, the proportion of both G 2/M and G 0/G 1 phase decreased. Positive transfection of ureB gene into SGC-7901 can change cell phenotype and induce cell apoptosis.