【作者】 赵雩卿；

【导师】 钱家鸣；

【作者基本信息】 中国协和医科大学 ， 消化内科， 2005， 博士

【摘要】 自幽门螺杆菌发现至今已有20多年，尽管对幽门螺杆菌及其相关疾病的研究已经取得了很大进展，但其感染造成慢性胃炎、萎缩性胃炎、消化性溃疡、胃癌及胃粘膜相关淋巴样组织淋巴瘤等不同临床结局的机制研究仍然是一个广为关注的热点。一般认为，导致不同疾病发生的原因除与宿主本身遗传特性和环境因素有关外，致病原的某些特性也是主要原因。宿主在不同毒力活性的菌株的作用下，胃肠上皮细胞是增殖还是凋亡、胃酸分泌水平是升高还是减低，都将直接导致不同的临床病理改变。 作为幽门螺杆菌主要毒力因子之一的空泡细胞毒素（VacA），因分泌量低、天然蛋白纯化困难、重组蛋白又缺乏毒力活性等原因，在其致病性的研究上远远落后于CagA。然而随着对幽门螺杆菌研究的深入，这种不需通过细菌直接粘附便可发挥毒性作用的分泌性蛋白又渐渐引起了研究者的兴趣。为此，本研究通过体外细胞实验，观察了从液体培养幽门螺杆菌上清液粗提的含有VacA的分泌蛋白对胃壁细胞酸分泌以及胃上皮细胞增殖／凋亡的调控作用。 1．液体培养VacA⁺幽门螺杆菌上清液粗提蛋白对离体兔胃壁细胞酸分泌的影响 本研究：1)应用液体培养方法培养幽门螺杆菌（NCTC 11637），以硫酸铵沉淀法提取菌液上清蛋白（BCF-P）并进行真空干燥浓缩，Hela细胞中性红摄取试验鉴定其空泡细胞活性。2)采用淘洗和连续密度梯度离心相结合的方法获得兔离体胃壁细胞，通过不同抑酸药物的作用，从丫叮橙染色、¹⁴C-AP摄取、H⁺／K⁺ATP酶α亚基mRNA表达水平三个方面来鉴定分离壁细胞具有酸分泌功能。3)电镜观察幽门螺杆菌上清液粗提蛋白对壁细胞形态学影响，并从¹⁴C-AP摄取、H⁺K⁺ATP酶mRNA表达水平两方面分析幽门螺杆菌上清液粗提蛋白及EGF对组胺刺激的壁细胞酸分泌的影响。 研究结果显示：1)液体培养幽门螺杆菌上清提取蛋白（BCF-P）中含有95KD空泡细胞毒素，对Hela细胞表现出空泡活性，中性红试验阳性。2)抗酸药物能显著抑制组胺刺激壁细胞对¹⁴C-AP的摄取（P＜0.05），并干扰基础状态下H⁺-K⁺ATP酶α亚基mRNA表达，提示淘洗分离获得的离体壁细胞具有良好的酸更多还原

【Abstract】 Since Helicobacter pylori (H.pylori) was found in 1982, the great development has been obtained in the study of H. pylori and its related diseases. However, the study on the mechanisms of different clinical outcomes (such as chronic gastritis, atrophic gastritis, peptic ulcer, stomach cancer and gastric mucosa-associated lymphoid tissue lymphoma) resulted from its infection is still a hot topic which has been attracted wide attention. It is generally agreed that not only do the different diseases caused by its infection depend on the hereditary characters of host and environmental factors, but also some features of the pathogen are the leading causes. Under the actions of strains with different virulence, whether gastrointestinal epithelial cells go into proliferation or apoptosis and whether the level of gastric acid secretion increases or decreases will directly lead to different clinical pathological changes.Vacuolating cytotoxin (VacA) is one of the leading virulent factors of Helicobacter pylori, but the research on its pathogenicity lags behind that of CagA because of its low secretory volume, difficult purification of its native protein and absence or change of virulence of its recombination protein. With the development of Helicobacter pylori research, this secretion protein, which can exert toxic action without the direct adhesion of bacteria, has attracted the interest of investigators little by little. For this reason, the purpose of this study was to observe the regulation and control of the crude extracted protein containing VacA of H.pylori Broth culture filtrate on the acid secretion of isolated parietal cells from rabbit and the proliferation/apoptosis of gastric epithelial cells. 1. The effect of the Broth culture filtrate protein of VacA~+ H. pylori on the acidsecretion of rabbit parietal cells in vitroThis study: 1) Helicobacter pylori (NCTC 11637, VacA~+) was grown in liquid更多还原