'Serous Cell secretion and Cystic Fibrosis Lung Disease'

囊性纤维化是由阴离子通道cftr基因突变引起的。CFTR、液体运输改变和导致大多数CF死亡的持续感染之间的联系尚未建立。我们将检验这一假设，即CF呼吸道疾病的发生，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是呼吸道浆液细胞顶膜上唯一的功能阴离子通道，因此它的缺失将减少丰富的浆液细胞的分泌。我们将通过比较慢性支气管炎患者和对照组患者呼吸道组织中的单腺体分泌物来直接检验这一假说。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。目标1的目的是通过使用单腺体监测来检验猪、羊和猫的腺体功能假说来量化和表征呼吸道粘膜下腺的分泌物。对于单个腺体，我们测量粘液分泌速度、离子含量、pH和跨皮细胞电位差，并收集未受污染的分泌物来测量粘弹性、固体含量和分泌的化合物，如溶菌酶。通过染料注射和随后的染色对单个腺体进行标记，以提供单腺体结构与功能的相关性。目的2的目的是通过使用上述方法比较正常对照、疾病对照和肺移植后CF受试者的气管和支气管的分泌物，来确定CF受试者的腺体分泌物是否减少或改变。我们预计在建议的5年捐赠期内将获得大约194个组织，包括37个CF、60个疾病控制组织和97个正常供体组织。AIMS 1和2中的实验设计和解释需要腺体分泌模型，实验结果验证了该模型。目标3的目的是利用开路Ussing小室和pH静态研究的CALU-3细胞模型，建立浆液细胞离子转运的综合模型。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。在目标4中，我们将利用这个‘虚拟腺体’来研究CALU-3细胞和原代培养的腺浆和腺粘液细胞的液体分泌。虚拟压盖在开路中运行，并可以识别流体传输的电子静音机制。这些方法的结合将使我们能够检验CF中腺体功能改变的主要假说，并将为其他呼吸道疾病中使用的气道腺功能提供新的信息。

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.