COLONIC CRYPT PERMEABILITY BARRIER IN CROHNS DISEASE

胃肠上皮的屏障功能使其对胃肠道具有很高的通透性。一些研究表明，肠道对大分子的通透性增加，这可能与克罗恩病的发病机制有关。胃肠道通透性的决定因素很复杂，而且还不完全清楚。我们已经在兔结肠隐窝细胞的顶端边界建立了一种不寻常的屏障，以阻止氨(一种弱碱)的渗透。根据临床研究人员奖K08 DKO2410，我们最近确定对弱酸二氧化碳(一种气体，如NH3)和丁酸也存在渗透屏障，所有这些都是结肠腔中丰富的发酵产物。这些分子中的每一个都具有中性亲脂形式，可以渗透到大多数细胞膜，影响细胞内的pH。事实上，使用最近开发的一种制备方法，我们发现极化的结肠表面细胞不同于它们位于隐窝中的祖细胞，对NH3、丁酸和二氧化碳具有很高的根尖渗透性。结肠腺对气体/亲脂分子的屏障对胃肠上皮的整体屏障功能可能是重要的。因此，重要的是要知道改变的顶端“小分子”屏障如何影响上皮细胞的健康，以及改变的小分子屏障是否先于和/或易导致与克罗恩病相关的“大分子”渗透性增加：由于结肠隐窝的不寻常的屏障功能直到最近才被认识到，目前还没有关于其与人类疾病的关系的数据。目前炎症性肠病的动物模型不能很好地代表最终导致人类克罗恩病的病理生理过程。幸运的是，我们可以在灌流的人体隐窝中研究隐窝细胞内的pH值。在最近的工作中，我们在所有患者的隐窝中观察到了NH3扩散的根尖屏障，但没有观察到NH4+的运输。此外，我们已经能够从动力学上描述组成人类隐窝的细胞之间的酸碱运输。这些技术进步应用于乙状结肠镜活检获得的隐窝，可以直接研究人类的小分子屏障，并能够评估改变的小分子屏障在克罗恩病发病机制中的作用。尽管根尖酸碱屏障现象的基础生物学研究在膜生物学中有着广泛的应用，但在这一应用中概述的研究长期集中在屏障的生物学和结肠细胞的pH与克罗恩病的关系上。

The barrier function of gastrointestinal epithelium confers high permselectivity to the GI tract. Several studies have demonstrated increased intestinal permeability to macromolecules that may be linked to the pathogenesis of Crohn's Disease. The determinants of gastrointestinal permeability are complex and incompletely understood. We have established the presence of an unusual barrier to the permeation of ammonia (a weak base) at the apical borders of rabbit colonic-crypt cells. Under Clinical Investigator Award K08 DKO2410, we recently determined that a permeability barrier exists as well to the weak acids CO2 (a gas, like NH3) and butyric acid, all of which are abundant products of fermentation in the colonic lumen. Each of these molecules possesses neutral lipophilic forms that permeate most cell membranes, affecting intracellular pH. Indeed, using a recently- developed preparation, we found that polarized colonic surface-cells, unlike their progenitors located in the crypt, possess high apical permeability to NH3, butyric acid, and CO2. The barrier to gases/lipophilic molecules in colonic glands is likely important to the overall barrier function of gastrointestinal epithelium. As such, it is important to know how altered apical "micromolecular" barriers affect epithelial cell health, and whether an altered micromolecular barrier precedes and/or predisposes to the increased "macromolecular" permeability associated with Crohn's Disease: As the unusual barrier function of colonic crypts has only recently been appreciated, no data as yet exist on its relationship to human disease. Present animal models of inflammatory bowel disease do not represent optimally the pathophysiologic processes that culminate in human Crohn's disease. Fortunately, we can study crypt cell intracellular pH in perfused human crypts. In recent work, we observed in human crypts an apical barrier to NH3 diffusion but not to NH4+ transport in all patients. In addition, we have been able to kinetically characterize acid-base transport among cells that comprise human crypts. These technical advances, applied to crypts obtained by sigmoidoscopic biopsy, permits direct study of micromolecular barriers in humans, and enables evaluation of the role of altered micromolecular barriers in the pathogenesis of Crohn's Disease. Although studies of the fundamental biology of apical acid-base barrier phenomenon have broad application to membrane biology, studies outlined in this application are focused long-term on the biology of the barrier and colonocyte pH as it relates to Crohn's Disease.