REGULATION OF RESPIRATORY TRACT FLUID LAYERS

呼吸道液层的调节

• 批准号: 6109984
• 负责人: Jonathan H Widdicombe
• 金额: $ 11.7万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6109984
• 关键词: Pseudomonas aeruginosa; X ray spectrometry; biological fluid transport; bradykinin; bronchial mucus; chloride channels; cystic fibrosis; electrophysiology; enzyme linked immunosorbent assay; fluorescence microscopy; hepatocyte growth factor; histamine; human tissue; ion transport; isoproterenol; lactoferrin; lysozyme; membrane permeability; methacholine; radiotracer; respiratory epithelium; secretion; sputum; synthetic peptide; tissue /cell culture

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.

显微镜显示气道内的液体由两种成分组成 层。纤毛周围有一层不同深度的粘液层 溶胶，一层覆盖纤毛的透明层。来源及监管 这些层尚未完全理解。然而，在囊性纤维化中， 据信表面上皮的异常离子传输会改变 这些层的关系或水合作用的方式会损害 粘膜纤毛间隙。在这个提案中，我们将测试两个假设。 首先，我们认为表面上皮的离子传输是 对调节纤毛周围溶胶的深度不太重要，这是 通过毛细作用力精确设定纤毛的长度 由较大的组合周向长度（每 cm2 600 m）产生 上皮表面）的纤毛。其次，我们提出有缺陷的 Cl 腺体分泌的液体主要负责最初的 囊性纤维化中粘液积聚。这些缺陷导致 脱水粘液不会从腺体开口完全分离 和/或不会被纤毛有效地运输。此外 腺体中 CFTR 的故障可能会改变硫酸化或其他特性 腺粘液，再次损害粘液纤毛清除。为了测试这些 假设，我们将研究渗透液和活性液如何流动 表面上皮改变粘液层的深度，以及这些如何改变 变化影响粘液运输。流体层将被可视化 扫描电子中快速冷冻组织的横截面 显微镜。我们将测量 CF 和介质诱导的液体分泌 使用电容探针技术进行非 CF 细胞培养，并从完整的 腺体通过微量移液器从导管开口取样。我们将确定是否 来自 CF 腺体和表面培养物的粘蛋白的硫酸化和流变学是 异常。最后，我们将确定本地化和总体水平 我们文化中的 CFTR，并与激素引起的变化相关 这些参数随着功能的变化而变化。其中大多数建议 实验将在我们最近开发的原代培养物上进行 表面和腺上皮细胞，显示出高水平的超微结构 和功能分化。