REGULATION OF RESPIRATORY TRACT FLUID LAYERS

显微镜检查显示，气道内的液体由两种 层次。睫状体周的顶部有一层深浅不一的粘液毯 溶胶，一层覆盖纤毛的透明层。的来源和调控 对这些层的理解并不完全。然而，在囊性纤维化中， 认为表面上皮细胞的异常离子转运改变了 这些层之间的关系或水合作用， 粘膜纤毛清除率在本提案中，我们将检验两个假设。 首先，我们认为表面上皮细胞的离子转运是 在调节睫状体周围溶胶的深度方面没有什么重要性， 通过毛细作用精确地固定在纤毛的长度上 由大的组合圆周长度（600 m/cm 2的 上皮表面）。其次，我们提出了亏损Cl 而腺体分泌的液体主要负责 囊性纤维化中粘液的积聚。这些缺陷导致 脱水粘液不会完全从腺体开口分离 和/或不能被纤毛有效地运输。此外 压盖中CFTR的故障可能会改变硫酸化或其他性质 腺体粘液，再次损害粘膜纤毛清除。测试这些 假设，我们将研究渗透和活性流体如何流过 表面上皮改变了粘液层的深度，以及这些 变化影响粘液运输。流体层将在 在扫描电子显微镜中快速冷冻组织的横截面 显微镜我们将测量跨CF的介质诱导的液体分泌， 非CF细胞培养物使用电容探针技术，并从完整的 通过从导管开口处微量吸管取样的腺体。我们将确定是否 来自CF腺和表面培养物粘蛋白的硫酸化和流变学， 不正常.最后，我们将确定本地化和总水平， CFTR在我们的文化，并相关的神经诱导的变化， 这些参数随功能的变化而变化。大多数建议 实验将在我们最近开发的原代培养物上进行 的表面和腺上皮，显示高水平的超微结构 功能分化。

Microscopy has shown the fluid lining the airways to consist of two layers. A mucous blanket of variable depth lies on top of the periciliary sol, a clear layer which bathes the cilia. The sources and regulation of these layers are incompletely understood. However, in cystic fibrosis, it is believed that abnormal ion transport by the surface epithelium alters the relationship or hydration of these layers in such a way as to impair mucociliary clearance. In this proposal we will test two hypotheses. Firstly, we suggest that ion transport by the surface epithelium is of little importance in regulating the depth of the periciliary sol, which is set precisely at the length of the cilia by forces of capillarity generated by the large combined circumferential length (600 m per cm2 of epithelial surface) of the cilia. Secondly, we propose that defective Cl and fluid secretion by the glands is mainly responsible for the initial accumulation of mucus in cystic fibrosis. These defects result in dehydrated mucus which will not detach fully from the gland openings and/or will not be effectively transported by the cilia. In addition malfunction of CFTR in glands may alter the sulfation or other properties of gland mucus, again impairing mucociliary clearance. To test these hypotheses, we will investigate how osmotic and active fluid flows across the surface epithelium alter the depth of the mucus layers, and how these changes affect mucus transport. The fluid layers will be visualized in transverse sections of rapidly frozen tissues in the Scanning electron microscope. We will measure mediator-induced fluid secretion across CF and non-CF cell cultures using a capacitance probe technique, and from intact glands by micropipette sampling from duct openings. We will determine if the sulfation and rheology of mucins from CF gland and surface cultures is abnormal. Finally, we will determine the localization and total levels of CFTR in our cultures, and correlate hormonally-induced alterations in these parameters with changes in function. Most of these proposed experiments will be performed on our recently developed primary cultures of surface and gland epithelia, which show high levels of ultrastructural and functional differentiation.