PPG -Airway Physiology and Pathophysiology in a Porcine CF Model

尽管CF的研究取得了进展，但我们仍然不了解呼吸道疾病的发病机制。一位少校 进展的障碍是缺乏除小鼠以外的CF动物模型。尽管CF小鼠已经被 它们被生产出来，不会患上通常在人类中发现的呼吸道疾病。因此，我们培育了一头猪 有针对性地破坏cftr基因。我们之所以选择猪，是因为它的肺有许多共同的解剖结构， 人类肺部的组织学、生化和生理学特征。在这个项目中，我们采用了独特的 有机会了解CFTR的缺失如何改变这一新的CF模型中的呼吸道上皮功能。几个 慢性阻塞性肺疾病气道上皮细胞电解质缺陷发病机制的假说 输送和异常呼吸道表面液体的体积和组成。这些主题是 我们的申请。通过与计划中的其他项目协作，我们将了解如何更改 上皮功能与炎症和感染有关，是该病的临床特征。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CFL肺的信息 这一次，然而，这正是CFTR缺失引发疾病的时候。 具体目的1.CFTR的缺失是否改变了猪的呼吸道上皮细胞的功能？我们将学习如何 用体外跨上皮电压测量方法研究CFTR缺失对体内离子转运的影响 分化的猪呼吸道上皮细胞的培养，新鲜切取的呼吸道上皮细胞，以及 肺部。我们还将了解心尖部Na+通道和替代CI“通道活动是否增加， 以及它们的功能与临床表型的关系。 特定目的2.猪CFTR的缺失是否改变了呼吸道表面液体(ASL)？争议很大 围绕着缺乏CFTR如何影响ASL的假设。它会降低ASL的音量吗？它会变吗？ ASL离子浓度？ASL酸碱度是否改变？在体外和体内都会发生变化吗？我们会回答 这些问题使用了几种独立的方法。通过研究小猪和动物在他们 发展成炎症和/或感染，我们将发现这些过程如何改变这种关键液体。 具体目标3.CFTR的缺失是否会扰乱粘液纤毛运输？缺少CFTR可能会导致缺陷 粘液纤毛运输(MCT)，从而引发呼吸道疾病。或者，其他因素可能会引发 MCT继发缺陷可能加重呼吸道疾病。当前数据不会 允许我们区分这些或其他假设。在这里，我们检验了CFTR丢失的假设 在体外和体内干扰正常的单核细胞移植。 这些研究将为CF的发病机制和病理生理提供新的见解，从而 加速发现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.