Mechanisms of H pylori-Induced Hypochlorhydria

描述(由申请人提供)：这项研究的目标是确定与幽门螺杆菌诱导的胃炎相关的低盐酸血症的分子机制。临床和体外证据表明，胃幽门螺杆菌感染与酸性分泌减少和炎症反应的产生有关。在很大一部分患者中，随之而来的胃炎发展为萎缩性胃炎、肠化生、不典型增生，最终发展为胃腺癌。本研究验证了幽门螺杆菌急性感染通过干扰HKα基因启动子上顺式调节元件与细胞反式激活蛋白的正常相互作用而下调H，K-ATPase(质子泵)基因表达的假说，从而抑制壁细胞酸的分泌。在初步研究中，外源H，K-ATPaseα亚单位(HKpha)启动子序列瞬时导入人胃 腺癌(AGS)细胞对酸性分泌激动剂和拮抗剂有反应，但受到幽门螺杆菌感染的抑制。这一方法为三个特定目的的实验奠定了基础：1)在胃上皮细胞中鉴定参与基因转录调控的幽门螺杆菌敏感的c/S调节元件和同源转录因子；2)研究特定的幽门螺杆菌基因型诱导HKa基因转录下调的机制； 3)研究幽门螺杆菌感染和未感染的人胃粘膜组织中Hpα亚单位基因的转录和翻译。通过缺失分析、凝胶迁移率漂移、超漂移和固相蛋白质/DNA相互作用分析，将研究基础的和幽门螺杆菌反应的顺式调节元件和反式激活蛋白。在Hp毒力相关基因缺失的突变株感染的ACTS细胞中，通过检测Hka启动子的活性，可以鉴定Hp的抑酸基因类型。最后，我们将在胃内pH值、感染状态、幽门螺杆菌菌株的特性和感染的解剖部位的背景下，通过实时RT-PCR和定量免疫化学在大量有充分记录的人类胃活检档案中测量胃H，K-ATPase mRNA水平和质子泵的表达。 这些研究将为幽门螺杆菌引起胃酸降低的机制奠定基础，并加深对幽门螺杆菌病理生理学的认识。

DESCRIPTION (provided by applicant): The goal of this study is to define the molecular mechanisms underlying the hypochlorhydria associated with Helicobacter pylori-induced gastritis. Clinical and in vitro evidence associates gastric corpus H. pylori infection with acid hyposecretion and generation of an inflammatory response. In a significant subset of patients, the ensuing gastritis progresses to atrophic gastritis, intestinal metaplasia, dysplasia, and eventually gastric adenocarcinoma. This study tests the hypothesis that acute H. pylori infection down-regulates H,K-ATPase (proton pump) gene expression by perturbing normal interactions of cis-regulatory elements on the HKalpha gene promoter and cellular trans-activating proteins, thereby inhibiting parietal cell acid secretion. In preliminary studies, exogenous H,K-ATPase alpha subunit (HKalpha) promoter sequences transiently transfected into human gastric adenocarcinoma (AGS) cells were responsive to acid secretory agonists and antagonists, and were inhibited by H. pylori infection. This approach forms the experimental basis for three Specific Aims: 1) To identify in gastric epithelial cells H. pylori-responsive c/s-regulatory elements and cognate transcription factors involved in regulation of HK( gene transcription; 2) To investigate the mechanisms whereby specific H. pylori genotypes induce down-regulation of HKalpha gene transcription; and 3) To investigate HKalpha subunit gene transcription and translation within H. pylori.infected and uninfected human gastric mucosa. Basal and H. pylori-responsive cis-regulatory elements and transactivating proteins in transfected ACTS cells will be investigated by deletion analysis, electrophoretic mobility shift, supershift, and solid-phase protein/DNA interaction assays. Acid-inhibitory H. pylori genotypes will be identified by assessing HKa promoter activity in transfected ACTS cells infected with H. pylori mutant strains deficient in virulence-associated genes. Finally, gastric H,K-ATPase mRNA levels and proton pump expression, as measured by real-time RT-PCR and quantitative immunochemistry in a large, well documented archive of human gastric biopsies, will be investigated in the context of intragastric pH, infection status, H. pylori strain identity, and anatomic site of infection. These studies will establish the mechanistic basis of H. pylori-induced gastric hypochlorhydria, and add to the understanding of H. pylori pathophysiology.