'Serous Cell secretion and Cystic Fibrosis Lung Disease'

“浆液细胞分泌与囊性纤维化肺病”

• 批准号: 6517405
• 负责人: JEFFREY J WINE
• 金额: $ 30.82万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-09-30 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6517405
• 关键词: acid base balance; acidity /alkalinity; animal tissue; bicarbonates; biological models; chloride channels; cystic fibrosis; enzyme linked immunosorbent assay; histopathology; human tissue; ion transport; lysozyme; membrane potentials; mucosal immunity; mucus; respiratory epithelium; secretion; tissue /cell culture; viscosity

Cystic fibrosis is caused by mutations in the gene for the anion channel CFTR. A link between CFTR, altered fluid transport and the persistent infections that cause most CF deaths has not been established. We will test the hypothesis that CF airways disease arises, at least in part, because of diminished or altered serous cell secretion. Our evidence is that CFTR is the only functional anion channel in the apical membranes of airway serous cells, so its loss will reduce rich serous cell secretion. We will directly test the hypothesis by comparing single gland secretions in airway tissues from CF and control humans. The series of experiments we propose have sufficient power to allow us to support or reject the serous cell dysfunction hypothesis. The proposal has 4 specific aims. The purpose of Aim 1 is to quantify and characterize airway submucosal gland secretions by using single gland monitoring to test hypotheses of gland function in pigs, sheep and cats. For individual glands, we measure the rate of mucus secretion, ion content, pH, and transepithelial potential difference and collect uncontaminated secretions to measure viscoelasticity, solids content, and secreted compounds such as lysozyme. Individual glands are labeled via dye injection and subsequent staining to provide single-gland, structure-function correlations. The goal of Aim 2 is to determine if gland secretions of CF subjects are diminished or altered by using the above methods to compare secretions from trachea and bronchi of normal control, disease control, and CF subjects, obtained following lung transplants. We project approximately 194 tissues will be acquired during the proposed 5-year grant period, consisting of 37 CF, 60 disease control, and 97 normal donor tissues. The design and interpretation of experiments in Aims 1 and 2 require a model of gland secretion, and the experimental results test the model. The purpose of Aim 3 is to develop a comprehensive model of ion transport for serous cells, using the Calu-3 cell model studied with open circuit Ussing chamber and pH stat studies. To link electrophysiological and fluid secretion studies, we have developed a novel method to study fluid secretion by epithelial cell sheets. In Aim 4, we will use this 'Virtual Gland' to study fluid secretion by Calu-3 cells and primary cultures of gland serous and gland mucous cells. The virtual gland operates in open circuit and can identify electrically silent mechanisms of fluid transport. The combination of methods will allow us to test the main hypothesis of altered gland function in CF, and will provide new information on airway gland function of use in other airway diseases.

囊性纤维化是由阴离子通道CFTR基因突变引起的。CFTR、体液转运改变和导致大多数CF死亡的持续性感染之间的联系尚未确定。我们将检验这一假设，即CF气道疾病的发生，至少部分是由于浆液细胞分泌减少或改变。我们的证据是CFTR是气道浆液细胞顶膜中唯一具有功能的阴离子通道，因此CFTR的缺失会减少丰富浆液细胞的分泌。我们将通过比较CF和对照人类气道组织中的单个腺体分泌物来直接验证这一假设。我们提出的一系列实验有足够的力量来支持或拒绝浆液细胞功能障碍假说。该提案有4个具体目标。Aim 1的目的是通过使用单个腺体监测来测试猪、羊和猫的腺体功能假设，量化和表征气道粘膜下腺分泌物。对于单个腺体，我们测量粘液分泌率、离子含量、pH值和经上皮电位差，并收集未污染的分泌物来测量粘弹性、固体含量和溶菌酶等分泌化合物。通过染料注射和随后的染色来标记单个腺体，以提供单个腺体的结构功能相关性。Aim 2的目的是通过使用上述方法比较肺移植后正常对照组、疾病对照组和CF患者的气管和支气管分泌物，以确定CF患者的腺体分泌物是否减少或改变。我们预计在5年资助期内将获得大约194个组织，包括37个CF组织，60个疾病控制组织和97个正常供体组织。Aims 1和Aims 2的实验设计和解释需要一个腺体分泌模型，实验结果验证了该模型。Aim 3的目的是建立一个全面的浆液细胞离子运输模型，使用开路使用室和pH stat研究的Calu-3细胞模型。为了将电生理和液体分泌研究联系起来，我们开发了一种新的方法来研究上皮细胞片的液体分泌。在Aim 4中，我们将使用这个“虚拟腺体”来研究Calu-3细胞和腺体浆液细胞和腺体粘液细胞的原代培养的液体分泌。虚拟腺体在开路运行，可以识别流体输送的电气静音机制。这些方法的结合将使我们能够验证CF中腺体功能改变的主要假设，并将为其他气道疾病的气道腺体功能提供新的信息。