幽门螺杆菌对胃黏膜上皮细胞自噬和凋亡的影响

Effect of Helicobacter pylori infection on autophagy and apoptosis of gastric epithelial cells

目的：探讨幽门螺杆菌（Hp）对胃黏膜上皮细胞（GES-1）自噬和凋亡的影响。方法：Hp与GES-1体外共培养，同时设立雷帕霉素（RAPA）、3-甲基腺嘌呤（3-MA）干预组及空白对照组，于2、4、6、12及24 h终止感染，通过免疫蛋白印迹、荧光染色、透射电镜、流式细胞术及细胞内活菌计数等方法观察Hp对GES-1自噬和凋亡的影响。结果：Hp＋GES-1组在2 h出现自噬标志蛋白LC3-Ⅱ表达及细胞内荧光自噬颗粒，透射电镜观察到细胞内出现双层或多层膜包裹的自噬小体样结构，细胞自噬在4 h达高峰，感染中后期逐渐减弱并消失；RAPA＋Hp＋GES-1组细胞自噬程度进一步增强，细胞凋亡率及细胞内活菌数均降低（P〈0.05-P〈0.01）；3-MA＋Hp＋GES-1组细胞自噬强度显著降低，而其细胞凋亡率和细胞内活菌数均明显升高（P 〈0.01）。结论：Hp可在感染早期诱导GES-1自噬；RAPA和3-MA可增强或降低自噬发生程度，从而减轻或促进细胞损伤。

Objective：To investigate the effects of Helicobacter pylori（Hp） on autophagy and apoptosis of gastric epithelial cells. Methods：Human gastric epithelial cell line GES-1（GES-1） were infected by Hp in vitro culture,the intervention groups of Hp-infected GES-1 were treated with autophagy inducer-rapamycin（RAPA） and autophagy inhibitor-3-methyladenine（3-MA）,and uninfected cell was set as the blank control group. Cells were collected at 2,4,6,12 and 24 h after infection. The effects of Hp infection on autophagy and apoptosis of GES-1 were observed by Western Blotting,fluorescent staining,transmission electron microscopy,flow cytometry and intracellular bacterial survival counting. Results：Autophagy marker protein microtubule associated protein 1 light chain 3-Ⅱexpression and cellular autophagy fluorescent particles were found at 2 h after treatment,the typical autophagic vacuoles with double or multilayer membrane in the cytoplasm were observed by transmission electron microscopy. Autophagy of infected cells peaked at 4 h, gradually decreased and disappeared. Autophagy of RAPA-treated cells infected by Hp further enhanced,while the apoptotic rate and intracellular bacterial survival were lower than those in RAPA-untreated infected cells（P〈0. 05 to P〈0. 01）. The autophagy of 3-MA-treated cells infected by Hp attenuated,and apoptosis and bacterial survival in this group were higher than those in RAPA-treated and -untreated infected cells（P〈0. 01）. Conclusions：Hp can induce the autophagy in gastric epithelial cells in the early infection. The autophagy inducer-RAPA and autophagy inhibitor-3-MA can promote or reduce the autophagy in Hp-infected cells,which can lessen or enhance injury of infected cells.