幽门螺杆菌cagA基因稳定转染胃上皮细胞及其对细胞的作用  

Effect of stable transfection of gastric epithelial cellls with recombinant of Helicobacter pylori cagA gene

作　　者：佘菲菲[1] 张静[2] 陈月秀[1] 陈豪[1] 

机构地区：[1]福建医科大学分子医学微生物学实验室,福州350004 [2]福建医科大学老年医学研究所

出　　处：《中国人兽共患病学报》2006年第1期29-32,共4页Chinese Journal of Zoonoses

基　　金：福建省自然科学基金重大项目(2001F003)

摘　　要：目的研究幽门螺杆菌(Hp)cagA基因重组子转染胃上皮细胞后对胃上皮细胞的作用。方法用PCR方法从Hp标准株NCTC11637中获取cagA全长基因,将其3.4kb的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-7901细胞,筛选耐潮霉素的细胞克隆,用RT-PCR方法检测细胞内cagA基因在转录水平的表达;用荧光染色技术、MTT、流式细胞术检测CagA对细胞表型、增殖、凋亡及细胞周期的影响。结果CagA阳性表达的细胞(ScagA)体积变大、胞浆出现空泡样变、胞膜出芽、细胞皱缩;用MTT法检测细胞增殖:ScagA细胞较SpcDNA3.1细胞(pcDNA3.1转染的细胞)生长增殖明显缓慢(P=0.009),表明ScagA细胞生长受抑制;流式细胞检测细胞凋亡结果:ScagA的凋亡率显著高于SpcD-NA3.1(P值:0.02);细胞周期分析显示:ScagA细胞有S期比率增高、G2-M、G0-G1期比率下降的趋势。结论cagA开放读码框架在培养细胞内的表达可引起细胞表型转变、抑制细胞增殖、促进细胞凋亡。To investigate the effect of stable trausfection of gastric epithelial cells with recombinant of Helicobacter pylori （Hp） cagA gene, the full length sequence of the cagA gene from the standard strain NCTC11637 of Hp was amplified by PCR, and its 3.4 kb open reading frame （ROF） of cagA gene was cloned into the eukaryotic expression vector pcDNA3.1. The recombinant thus obtained was used to transfect the gastric carcinoma cell line SCC-7901 cells, and the hygromycin-resistant clone was screened. Meanwhile, the transcription level expression of cagA gene was assayed by RT-PCR, and the influences of CagA protein on the phenotypes, proliferation, apoptosis of cells and cell cycles were observed by means of fluorescence staining, MTT assay and flow cytometry respectively. It was found that the size of the positive CagA-expressing cells appeared to be larger with vacuolar changes in cytoplasm, membrane budding and cell shrinkage. As demonstrated by the MTT assay, the growth rate of the CagA-expressing cells was much slower than that of cells transfeeted with pcDNA3.1, indicating the specific inhibition of growth of the CagA expressing cells. However, the apoptotic rate of the CagA-expressing cells was somewhat higher than that of cells transfected with pcDNA3.1 as demonstrated by flow cytometry. Analysis of the cell cycle showed that in the Cag-expressing cells, the proportion of the S-phase increased, but that of both G2/M and G0/G1decreased. It is evident that the transfection of gastric epithelial cells with recombinant of H. pylori cagA gene can induce the changes in cell types, inhibition of cell proliferation and cell apoptosis in vitro.

关 键 词：幽门螺杆菌 CAGA 细胞增殖 细胞凋亡 基因转染 

分 类 号：R378[医药卫生—病原生物学]