幽门螺杆菌对胃黏膜上皮细胞自噬和凋亡的影响

摘要

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.%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。