摘要
目的探究幽门螺杆菌(H.pylori)的细胞毒素相关基因A蛋白(CagA)过表达对胃癌细胞葡萄糖转运蛋白1(GLUT1)表达的影响。方法用过表达cagA腺病毒感染人胃癌细胞株AGS和人原代胃癌细胞,免疫荧光检测GLUT1的表达;用过表达glut1慢病毒及其空载体慢病毒感染胃癌细胞株AGS和BGC-823、0.002 mg/L嘌呤霉素筛选,用Western blot、免疫荧光验证稳转细胞株;最后用过表达cagA腺病毒感染过表达GLUT1的稳转细胞株,免疫荧光检测GLUT1与CagA的细胞定位。结果成功建立过表达glut1的稳转细胞株AGS/glut1和BGC-823/glut1,免疫荧光结果显示CagA和GLUT1蛋白主要在细胞膜上表达,且过表达CagA能引起细胞膜上GLUT1蛋白达降低并出现碎片化,且CagA与Glut1在细胞膜上共定位。结论CagA与GLUT1共定位于细胞膜上,破坏细胞膜,抑制GLUT1蛋白的表达。
Objective To study the effect of overexpression of Helicobacter pylori(H.pylori)Cytotoxin associated gene A(CagA)on glucose transporter 1(GLUT1)expression in gastric cancer cells.Methods Human gastric cancer cell line AGS cells and primary gastric cancer cells were infected with the cagA-overexpressing adenovirus,GLUT1 protein expression was detected by immunofluorescence.Next,gastric cancer cell lines AGS and BGC-823 were infected with the glut1-overexpressing lentivirus and its empty vector lentivirus,and then screened by using 0.002 mg/L puromycin.The stable cell lines were verified by Western blot and immunofluorescence.Finally,the stable cell lines overexpressing glut1 were infected with the adenovirus carrying cagA.The subcellular localization of GLUT1 and CagA was detected using immunofluorescence.Results The stable cell lines AGS/glut1 and BGC-823/glut1 which overexpressed glut1 were successfully established.The immunofluorescence results showed that CagA and GLUT1 protein were mainly expressed on the cell membrane of gastric cancer cells.The CagA overexpression decreased GLUT1 protein expression and fragmented GLUT1.Confocal microscope results showed the co-localization of CagA with GLUT1 on the cell membrane of gastric cancer cells.Conclusion CagA is co-localized with GLUT1 on the cell membrane,damaging the cell membrane and inhibiting the expression of GLUT1 protein.
作者
程薇
杨丽萍
周建奖
谢渊
全欣莹
陈定宇
王琴容
赵艳
CHENG Wei;YANG Liping;ZHOU Jianjiang;XIE Yuan;QUAN Xinying;CHEN Dingyu;WANG Qinrong;ZHAO Yan(Education Ministry Key Laboratory of Endemic and Minority Diseases&Key Laboratory of Molecular Biology of Guizhou Medical University,Guiyang 550004,Guizhou,China;Clinical Medicine Research Center,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)
出处
《贵州医科大学学报》
CAS
2021年第4期381-386,共6页
Journal of Guizhou Medical University
基金
国家自然科学基金(31760328)
贵州省科技计划项目黔科合支撑[(2020)4Y026]
贵州省科技计划项目黔科合基础[(2020)1Y333]。
关键词
胃肿瘤
螺杆菌
幽门
细胞毒素相关基因A蛋白
葡萄糖转运蛋白1
stomach neoplasms
Helicobacter pylori
cytotoxin-related gene A protein(CagA)
glucose transporter 1(GLUT1)
