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）的缺失如何影响硫酸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。高水平的硫酸糖合物和异常的翻译后加工被认为是导致胰腺和肠道阻塞的原因，CF中营养物质的消化和吸收异常。模型黏液样硫酸糖蛋白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，Muclin在这些组织中的免疫定位表明，它与腺泡管腔的蛋白栓和肠隐窝管腔的粘蛋白栓有关。假设是，在缺乏功能性CFTR的情况下，粘蛋白样糖缀合物，如Muclin，会过度表达并改变翻译后加工。然后，它们在胰腺腺泡和小肠隐窝的腔内造成有害的蛋白质聚集体积累。将讨论以下具体目标。1. 验证在正常表达CFTR的上皮细胞即肠隐窝细胞中，CFTR缺失时Muclin翻译后加工发生改变的假设，并确定这种改变的机制。测定正常肠和CF肠Muclin的碳水化合物结构和蛋白核大小。2. 验证过量Muclin改变CF小鼠胃肠道组织蛋白分泌途径的假说。我们将在体内和体外研究正常小鼠和CF小鼠的胰腺分泌物，并评估pH对Muclin与酵母菌相互作用的影响。同时测量CF和正常细胞分泌腔室的腔内pH值，以检测CFTR是否对细胞内pH梯度有功能作用。3. 验证肠酸性异常介导CF发病及Muclin过表达的假说。我们将比较CF小鼠的病理和Muclin的表达，并将CF小鼠与胃泌素缺乏小鼠（胃酸分泌不足）杂交以使肠道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.