PPG -Airway Physiology and Pathophysiology in a Porcine CF Model

尽管CF研究取得了进展，但我们仍然不了解气道疾病的发病机制。一个主要 进展的障碍是缺乏除小鼠以外的CF动物模型。虽然CF小鼠已经被 它们不会产生通常在人类中发现的呼吸道疾病。因此，我们开发了一种猪 有针对性地破坏CFTR基因我们选择猪是因为它的肺在解剖学上， 组织学、生物化学和生理学特征。在这个项目中，我们采取了独特的 在这种新的CF模型中，有机会了解CFTR的丧失如何改变气道上皮功能。几 关于CF气道疾病的发病机制的假说以气道上皮电解质缺陷为中心 运输和异常气道表面液体量和组成。这些主题是 我们的申请通过与该计划中其他项目的合作，我们将了解如何改变 上皮功能与炎症和感染有关，炎症和感染是该疾病的临床标志。我们专注于 早期出生后和年轻的猪，因为严重缺乏关于人类CF肺的信息， 这一次，而这正是CFTR缺失引发疾病的时候。 具体目标1。CFTR缺失是否改变猪气道上皮细胞的功能？我们将学习如何 缺乏CFTR改变离子转运在体内使用跨上皮电压的测量，在体外使用 在新鲜切除的气道上皮中，以及在猪的远端气道中， 肺我们还将了解顶端Na+通道和替代Cl-通道活动是否增加， 以及它们的功能与临床表型的关系。 具体目标2。猪CFTR丢失是否会改变气道表面液体（ASL）？很大争议 围绕着缺乏CFTR如何影响ASL的假设。是否会减少ASL音量？它会改变吗 ASL离子浓度？ASL pH值是否改变？在体外和体内是否都发生了变化？我们将回答 这些问题使用几个独立的方法。通过研究小猪和动物， 当炎症和/或感染发生时，我们将发现这些过程如何改变这种重要的液体。 具体目标3。CFTR的缺失会破坏粘膜纤毛的转运吗？缺乏CFTR可能导致缺陷 粘膜纤毛转运（MCT），从而引发气道疾病。或者，其他因素可能会引发 疾病过程中，然后在MCT继发性缺陷可能会加重气道疾病。当前数据不 让我们区分这些或其他假设。在这里，我们测试的假设，损失CFTR 在体外和体内破坏正常的MCT。 这些研究将为CF发病机制和病理生理学提供新的见解， 加速发现治疗这种致命疾病的新疗法。

Despite advances in CF research, we still do not understand the pathogenesis of airways disease. A major mpediment to progress is lack of a CF animal model other than the mouse. Although CF mice have been produced, they do not develop the airway disease typically found in humans. Therefore, we developed a pig with a targeted disruption of the CFTR gene. We chose the pig because its lungs share many anatomical, histological, biochemical, and physiologic features with human lungs. In this project, we take the unique opportunity to learn how loss of CFTR alters airway epithelial function in this new CF model. Several hypotheses about the pathogenesis of CF airway disease center on defective airway epithelial electrolyte transport and abnormal airway surface liquid volume and composition. These topics are the main focus of our application. Through collaborations with the other projects in the Program, we will discern how altered epithelial function relates to inflammation and infection, clinical hallmarks of the disease. We concentrate on early postnatal and young pigs because there is a critical lack of information about the human CF lung during this time, and yet this is precisely when loss of CFTR initiates disease. Specific Aim 1. Does loss of CFTR alter the function of porcine airway epithelia? We will learn how lack of CFTR changes ion transport in vivo using measurements of transepithelial voltage, in vitro using cultures of differentiated pig airway epithelia, in freshly excised airway epithelia, and in the distal airways of the lung. We will also learn whether apical Na+ channel and alternative CI" channel activities are increased, and how their function relates to the clinical phenotype. Specific Aim 2. Does loss of porcine CFTR change the airway surface liquid (ASL)? Much controversy surrounds hypotheses about how lack of CFTR affects ASL. Does it reduce ASL volume? Does it change ASL ion concentrations? Is ASL pH altered? Do changes occur both in vitro and in vivo? We will answer these questions using several independent methods. By studying both young pigs and animals after they develop inflammation and/or infection, we will discover how these processes change this critical liquid. Specific Aim 3. Does loss of CFTR disrupt mucociliary transport? Lack of CFTR might cause defective mucociliary transport (MCT) thereby initiating airway disease. Alternatively, other factors might initiate the disease process, and then secondary defects in MCT might worsen airways disease. Current data do not allow us to discriminate between these or other hypotheses. Here, we test the hypothesis that loss of CFTR disrupts normal MCT in vitro and in vivo. These studies will provide new insights into both CF pathogenesis and pathophysiology and thereby accelerate the discovery of novel therapies for this lethal disease.