幽门螺杆菌体外诱导大鼠胃黏膜上皮细胞凋亡

Helicobacter pylori induces apoptosis of rat gastric epithelial cells in vitro

目的:研究幽门螺杆菌(Hpylori)在大鼠胃黏膜上皮诱导细胞凋亡中的作用,并初步探讨其中凋亡相关基因表达的情况,为胃癌发病机制提供依据.方法:Hpylori超声提取液来自SydneySS-1Hpylori菌株.大鼠胃黏膜细胞OUMS-37为永生化细胞,当细胞生长至60%融合时,加入不同浓度的Hpylori超声提取液,同时设置空白,于培养的24-48h收集细胞进行形态观察.Westhernblotting检测P53蛋白表达,Northernblotting检测bax、bcl-2mRNA的表达.结果:细胞经Hpylori作用48h后在高倍镜下观察到细胞核碎裂成大小不等的块状,表现出细胞凋亡如细胞皱缩,胞浆嗜碱性,核染色质固缩致密,核染色质断裂,形成大小不等的胞内核小体,部分细胞核膜消失,核染色质聚集中细胞中央,呈现分裂期的形态学等形态学特征,对照组未出现以上特征性改变.培养细胞经过Hpylori作用后提取DNA,经15g/L琼脂糖凝胶电泳,在紫外线灯下观察呈现不连续的梯状结构电泳条带.培养细胞经过Hpylori作用后,Westhernblotting显示P53蛋白表达随Hpylori超声提取液浓度而升高,Northernblotting显示baxmRNA表达随Hpylori浓度而增加,bcl-2mRNA表达随Hpylori浓度而降低.结论:Hpylori超声提取液可在体外诱导鼠胃黏膜上皮细胞凋亡.其机制可能通过上调野生型P53蛋白和凋亡促进基因baxmRNA表达,并下调凋亡抑制基因bcl-2mRNA表达.提示Hpylori感染可通过干扰胃上皮细胞增殖与凋亡之间的平衡在胃癌病因学中发挥作用.

AIM： To investigate the effect of Helicobacter pylori sonicated extract on the apoptosis of rat gastric epithelial cells as well as the expression of apoptosis-related genes in vitro. METHODS： H pylori sonicated extract from strain Sydney SS-1 was cultured with OUMS-37, a kind of immortalized rat gastric cell lines. Apoptosis of the cells was confirmed according to specific changes of morphology and DNA ladder 24-48 h after co-incubation. The expression of P53 protein was detected by Western blotting and the expression ofbax and bcl-2 mRNA were observed by Northern blotting. RESULTS： The specific morphology of the cells such as shrinkage, condensation, margination of nuclear chromatin and apoptotic bodies were observed under light microscope. DNA ladder was manifested by fragment analysis, Western blotting showed a dose-dependent increased expression of wild-type P53 protein and Northern blotting showed a dose-pendent increased expression of bax mRNA and reduced expression of bcl-2 mRNA in the treated cells. CONCLUSION： Hpylon sonicated extract induces the apoptosis in vitro through up-regulation of wild-type P53 protein and bax mRNA expression, and down-regulation of bcl-2 mRNA expression, suggesting that H pylon infection may interrupt the balance between proliferation and apoptosis of the gastric epithelial cells, which plays a key role in gastric carcinogenesis.