Repairing pre-epithelial defenses after gastric damage

描述(申请人提供)：胃必须通过胃上皮细胞分泌的HCl。这一挑战因化合物(如非类固醇抗炎药)或生物体(幽门螺杆菌)的频繁存在而加剧--它们损害胃对酸的屏障，当屏障被持续突破时会导致溃疡。碱性粘膜旁凝胶层是胃防御的第一道防线：起到保护胃上皮细胞免受胃腔酸性物质反向扩散的作用。我们开发了一种体内共聚焦显微镜方法来非侵入性地测量上皮前隔室的pH值，并将该方法扩展到包括使用双光子光吸收来在几秒钟内致命地损伤选定的表面上皮细胞。作为回应，组织在粘膜旁层表现出大量但短暂的碱液涌动，产生一个粘液帽来覆盖脱落细胞的“微损伤”，并在几分钟内完成上皮修复。这是第一个由于上皮更新或摄入的食物对上皮的物理损伤而必须每天发生的微观上皮破坏的模型。我们的目标是确定限制胃损伤进展的因素，并促进显微损伤后上皮和粘膜旁防御的恢复，使用共焦显微镜的微损伤模型实时跟踪上皮和粘膜前修复。我们的第一个目标是询问表面pH调节是否对急性胃损伤和修复的进展很重要。利用野生型小鼠，我们将测试短暂的碱浪涌是否对上皮细胞具有保护作用，并在修复过程中定位修复的上皮屏障的位置。利用COX基因敲除小鼠和COX抑制剂，我们将确定哪些环氧合酶亚型调节急性损伤的组织修复。第二个目的是研究动素在上皮前和上皮性防御中的作用。使用野生型和运动原基因缺失的小鼠(TFF1、TFF2和转化生长因子-β1基因敲除)，我们将询问运动原是否调节健康组织的表面pH，或刺激微小病变后的上皮修复。固定的标本将被用来询问运动素是否额外作用于微病变附近的细胞以抑制增殖或抑制凋亡。在第三个目标中，我们将询问是否可以在微小病变上开始更广泛的幽门螺杆菌损害。我们将测试微小病变是否在最初定植期间引导幽门螺杆菌进入胃表面，以及幽门螺杆菌诱导的胃炎是否影响对局灶性上皮病变的反应。结果将扩展我们关于加强或削弱胃防御的条件的知识，以应对在正常的上皮更新过程中每天遇到的大小的浅表病变。

DESCRIPTION (provided by applicant): The stomach must survive HCl secreted by the gastric epithelium. This challenge is heightened by the frequent presence of compounds (e.g. non-steroidal anti-inflammatory drugs) or organisms (Helicobacter pylori) -which compromise the gastric barrier to acid, leading to ulcers when the barrier is breached for sustained intervals. An alkaline juxtamucosal gel layer is the first line of gastric defense: acting to protect the gastric epithelium from back-diffusion of acid from the lumen. We developed an in vivo confocal microscopy approach to non-invasively measure pH in the pre-epithelial compartment, and have extended this methodology to include use of two-photon light absorption to fatally damage selected surface epithelial cells in a few seconds. In response, the tissue manifests a large but transient surge of alkali in the juxtamucosal layer, creates a mucoid cap to cover the "micro-lesion" of exfoliated cells, and undergoes epithelial restitution in a matter of minutes. This is the first model of the microscopic epithelial disruptions that must occur daily due to epithelial renewal, or physical injury of the epithelium by ingested food stuffs. Our objective is to define the elements limiting progression of gastric damage and favoring restoration of epithelial and juxtamucosal defenses after microscopic lesions, using the micro-lesion model with confocal microscopy to follow epithelial and pre-epitheila repair in real-time. Our first aim asks if surface pH regulation is important to the progression, of acute gastric damage and repair. Using wild-type mice, we will test if the transient alkali surge is protective to the epithelium, and localize the site of the restored epithelial barrier during repair. Using COX-knockout mice and COX inhibitors, we will define which cycloxygenase isoforms regulate tissue repair from acute damage. The second aim examines the roles of motogens in pre-epithelial and epithelial defense. Using wild-type and motogen null mice (TFF1, TFF2 and TGF-beta1 knockouts) we will ask if motogens regulate surface pH in healthy tissue, or stimulate epithelial restitution after micro-lesions. Fixed specimens will be used to ask if motogens additionally act on cells adjacent to the micro-lesion to inhibit proliferation or inhibit apoptosis. In the third aim, we will ask if more extensive damage caused by H. pylori can be initiated on micro-lesions. We will test if micro-lesions are an attractant guiding H. pylori to the gastric surface during initial colonization, and if H. pylori-induced gastritis affects the response to focal epithelial lesions. Results will extend our knowledge about conditions that strengthen or weaken gastric defense, in response to a superficial lesion of a size that is encountered daily during normal epithelial renewal.