PATHOGENEISIS OF CYSTIC FIBROSIS IN THE GI SYSTEM

长期目标是了解功能性CFTR（囊性纤维化跨膜调节因子）的丧失如何影响硫酸化糖缀合物的表达和翻译后加工以及这些改变的糖蛋白在囊性纤维化（CF）发展中的作用。高水平的硫酸化糖缀合物与异常的翻译后加工被认为有助于胰腺和肠中的阻塞，并且营养物的消化和吸收在CF中是异常的。模型粘蛋白样硫酸化糖蛋白Muclin将用于探索这些问题。Muclin在CFTR（-/-）小鼠的胰腺和小肠中过表达，并且Muclin在这些组织中的免疫定位显示其与腺泡腔中的蛋白质塞和肠隐窝腔中的粘蛋白塞相关。假设是在功能性CFTR不存在的情况下，粘蛋白样糖缀合物（例如Muclin）过表达并且改变了翻译后加工。然后，它们导致蛋白质聚集体在胰腺腺泡和小肠隐窝的腔中的有害积累。将处理以下具体目标。1.为了检验以下假设：在正常表达CFTR的上皮细胞中，Muclin的翻译后加工在CFTR不存在的情况下发生改变，即，肠腺细胞，并确定这种变化的机制。将测定正常和CF肠中Muclin的碳水化合物结构和蛋白质核心大小。2.验证过量Muclin改变CF小鼠胃肠道组织中蛋白质分泌途径的假设。将在正常和CF小鼠体内和体外研究胰腺分泌物，并评估pH值对Muclin与酶原相互作用的影响。此外，将测量CF和正常细胞中分泌区室的腔pH，以测试CFTR是否在细胞中的pH梯度中具有功能作用。3.检验肠道酸度异常介导CF发病机制和Muclin过度表达的假设。将在CF小鼠中比较病理学和Muclin表达，并将CF小鼠与胃泌素缺陷小鼠（缺乏胃酸分泌）杂交以使肠PH正常化。

The long-term objective is to understand how loss of functional CFTR (the cystic fibrosis transmembrane regulator) affects expression and post- translational processing of sulfated glycoconjugates and the role of these altered glycoproteins in the development of cystic fibrosis (CF). High levels of sulfated glycoconjugates with abnormal post-translational processing are believed to contribute to obstruction in the pancreas and intestines, and digestion and absorption of nutrients is abnormal in CF. The model mucin-like sulfated glycoprotein Muclin will be used to explore these issues. Muclin is over-expressed in the pancreas and small intestine of CFTR (-/-) mice, and immunolocalization of Muclin in these tissues shows that it is associated with protein plugs in the acinar lumen and mucin plugs in the intestinal crypt lumen. The hypothesis is that in the absence of functional CFTR, mucin-like glycoconjugates, such as Muclin, are over-expressed and have altered post-translational processing. They then contribute to the deleterious accumulation of protein aggregates in the lumina of the pancreatic acini and small intestinal crypts. The following specific aims will be addressed. 1. To test the hypothesis that post-translational processing of Muclin is altered in the absence of CFTR in epithelial cells which normally express CFTR, i.e., the intestinal crypt cell, and to determine the mechanism of this change. The carbohydrate structure and protein core size of Muclin in normal and CF intestine will be determined. 2. To test the hypothesis that excess Muclin alters the protein secretory pathway in gastrointestinal tissues of CF mice. Pancreatic secretions will be studied in vivo and in vitro in normal and CF mice, and the effect of pH on the interaction of Muclin with zymogens will be assessed. Also, the luminal pH of secretory compartments in CF and normal cells will be measured to test whether CFTR has a functional role in pH gradients in the cell. 3. To test the hypothesis that abnormal acidity in the intestine mediates CF pathogenesis and the over-expression of Muclin. Pathology and Muclin expression will be compared in CF mice, and CF mice crossed with gastrin deficient mice (lack gastric acid secretion) to normalize intestinal Ph.