VP3和TRAIL基因共表达沙门菌载体对胃癌细胞生长作用的影响

Effects of co-expression of VP3 and TRAIL in Salmonella typhimurium vector on the growth of gastric cancer cell lines

目的:构建鸡贫血病毒(chicken anemia virus,CAV)VP3基因和肿瘤坏死因子相关凋亡诱导配体(TNF-related apopto-sis-inducing ligand,TRAIL)基因的真核表达载体,并观察其对胃癌细胞生长的影响情况。方法:采用分子克隆技术,将VP3基因插入真核表达载体pBud-TRAIL中,构建获得质粒pBud-TRAIL-VP3,采用电转化法将该重组质粒转入减毒沙门菌SL7207,再直接转染胃癌细胞株SGC-7901。48h后采用RT-PCR检测VP3基因表达状况并在荧光显微镜下观察细胞内有无绿色荧光蛋白(green fluorescent protein,GFP)表达。MTT法检测重组菌株对SGC-7901细胞生长状况的影响。AO/EB荧光染色检测其对肿瘤细胞凋亡率的影响。透射电镜观察胃癌细胞形态改变。结果:获得VP3基因正确插入的真核表达载体;重组质粒经减毒沙门菌介导转染胃癌细胞后,RT-PCR检测到VP3基因存在;荧光显微镜下观察到在转染细胞内有GFP表达;转染48h后可见到TRAIL和VP3对胃癌细胞生长有明显抑制作用(P<0.05),荧光染色可见胃癌细胞有凋亡现象,pBud-TRAIL-VP3能够明显提高胃癌细胞的凋亡率(P<0.05),透射电镜观察到转染细胞发生凋亡形态变化。结论:减毒沙门菌可将外源基因VP3和TRAIL基因导入胃癌细胞并进行表达,TRAIL和VP3对胃癌细胞的生长起协同抑制作用并能促进胃癌细胞的凋亡。

Objective：To construct an eukaryotic expression vector carrying chicken anemia virus （ VP3 ） gene and TNF-related apoptosis-inducing ligand （TRAIL） gene, and study its inhibitory effect on gastric cancer cell lines. Methods： VP3 gene was inserted into the expression vector of pBud-TRAIL by molecular coloning technology. The plasmid pBud-TRAIL-VP3 was transformed into attenuated Salmonella typhimurium by electric transformation method and directly transfected into gastric cancer cell lines SGC-7901. The VP3 gene expression was detected by RT-PCR at 48 h after transfection and the expression of green fluorescence protein （GFP） was observed under microscope. The proliferation of gastric cancer cells was assessed by MTT assay. Apoptosis was determined by using AO/EB fluorescence staining. Morphological changes of gastric cancer cells were observed under transmission electron microscope. Results： VP3 gene was correctly inserted into eukaryotic expression vector. We detected the existence of VP3 gene in gastric cancer cells after transfection of the plasmid mediated by attenuated Salmonella typhimurium by RT-PCR. We observed the expression of enhanced green fluorescent protein （EGFP） with fluorescent microscopy after the recombinant plasmid was transfected into gastric cells. The recombinant plasmid significantly inhibited the growth of gastric cancer cells （P 〈 0.05 ）. AO/EB fluorescence staining indicated transfection of pBud-TRAIL-VP3 markedly increased the apoptotic rate of gastric cancer cells （P 〈 0.05 ）. Transfected cells presented typical morphologic features of apoptosis under transmission electron microscope. Conclusion： VP3 gene and TRAIL gene could be introduced into gastric cancer cells by attenuated Salmonella typhimurium and had stable expressions. They had synergistic effects in inhibi- ting the proliferation and inducing apoptosis of gastric cancer cells.