Molecular Analysis of Airway Secretory Cells in Health and Disease

慢性阻塞性肺病（COPD）是美国和世界范围内的第三大死亡原因， 并且它仍然是 疾病 无法治愈的部分原因是对其细胞机制的不完全理解。小气道 (SAD)和肺气肿是COPD的重要病理表型。SAD的特点是 在小气道（直径<2mm）中产生过多的粘液和减少的肺泡附着。伤心 也可能恶化肺气肿，肺泡组织的逐渐破坏。然而，机制 小气道粘蛋白过度产生和肺泡破坏的发病机制尚不清楚 明白重要的是，粘蛋白是由上皮祖细胞，俱乐部细胞，它可以分化成 肺泡II型细胞用于组织修复。在这里，我们已经发现，聚合物凝胶形成的过度生产 粘蛋白MUC5B可能与小鼠COPD模型中俱乐部细胞导致的肺泡再生受损有关。因此，我们认为， 这项建议旨在检验中心假设，即健康人的俱乐部细胞维持和修复肺泡 COPD依赖于MUC5B的表达。具体目标1将检验MUC5B 表达抑制了在体内平衡期间俱乐部细胞向ATII细胞的分化。我会用类器官分析 为了测试来自健康小鼠的MUC5B表达和非表达俱乐部细胞的稳态祖细胞功能 用他莫昔芬诱导的俱乐部细胞谱系追踪和内源性MUC5B的荧光标记。进一步 研究将使用具有Muc5b的遗传缺陷或转基因过表达的小鼠。第2章测试 在COPD小鼠模型中MUC5B的过度产生抑制肺泡修复的假设。来自Aim的老鼠 1例将用猪胰弹性蛋白酶（PPE）处理，以测试俱乐部细胞中过量的MUC5B是否损害 体外和体内的肺泡分化，以及Muc5b的条件性消耗是否改善SAD和 肺气肿最后，具体目标3将测试干扰刺激粘蛋白过度产生的假设， 通过恢复俱乐部细胞的祖细胞功能来预防SAD和肺气肿。我将研究 细胞外与细胞内过量MUC5B如何损害俱乐部细胞的祖细胞功能，我将确定 调节俱乐部细胞粘液和祖细胞功能的新基因， 人COPD。这些研究将填补我们对小细胞中俱乐部细胞的异质性功能的知识空白。 气道内稳态和疾病。目标1、2和目标3的部分将在K99训练阶段完成。 我训练的关键要素将是学习粘蛋白生物学和生物信息学的新领域，这将帮助我 发展我的职业生涯作为一个转化COPD研究人员。我的学习成果将为我的将来打下坚实的基础。 一系列关于COPD中调节上皮表型的途径的独立研究和新的 R00期SAD和肺气肿的治疗药物。凭借我出色的指导团队和 特殊的培训环境，我理想的定位，以实现拟议的科学培训和职业生涯 发展目标，并成熟为独立调查员。

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the US and worldwide, and it remains disease incurable in part due to an incomplete understanding of its cellular mechanisms. Small airway (SAD) and emphysema are important pathological phenotypes of COPD. SAD is characterized by excessive mucus production and reduced alveolar attachment in the small airways (<2mm in diameter). SAD may also worsen emphysema, the progressive destruction of alveolar tissue. However, the mechanisms underlying the pathogenesis of small airway mucin overproduction and alveolar destruction are not well understood. Importantly, mucin is produced by an epithelial progenitor, the club cell, which can differentiate into alveolar type II cells for tissue repair. Here, we have discovered that overproduction of a polymeric gel-forming mucin MUC5B may be linked to impaired alveolar regeneration by club cells in a murine COPD model. Therefore, this proposal aims to test the central hypothesis that alveolar maintenance and repair by club cells in health and COPD is dependent on MUC5B expression. Specific Aim 1 will test the hypothesis that MUC5B expression inhibits differentiation of club cells into ATII cells during homeostasis. I will employ organoid assays to test steady state progenitor functions of MUC5B-expressing and non-expressing club cells from healthy mice with tamoxifen-induced lineage tracing of club cells and fluorescent labeling of endogenous MUC5B. Further studies will employ mice with genetic deficiency or transgenic overexpression of Muc5b. Specific Aim 2 will test the hypothesis that overproduction of MUC5B inhibits alveolar repair in a mouse model of COPD. Mice from Aim 1 will be treated with porcine pancreatic elastase (PPE) to test whether excessive MUC5B in club cells impairs alveolar differentiation in vitro and in vivo, and whether conditional depletion of Muc5b ameliorates SAD and emphysema. Finally, Specific Aim 3 will test the hypothesis that disrupting stimulated mucin overproduction prevents SAD and emphysema by restoring progenitor function of club cells. I will investigate the mechanism of how extra- versus intra-cellular excessive MUC5B impairs progenitor functions of club cells, and I will identify novel genes that regulate club cell mucous and progenitor functions and expand on translational studies in human COPD. These studies will fill gaps in our knowledge of heterogeneous functions of club cells in small airways in homeostasis and disease. Aim 1, 2 and part of Aim 3 will be completed during the K99 training phase. Key elements of my training will be learning new fields of mucin biology and bioinformatics that will help me develop my career as a translational COPD researcher. Results of my studies will supply a foundation for strong lines of independent research on pathways regulating epithelial phenotypes in COPD and development of novel therapeutics for SAD and emphysema in the R00 phase. With my outstanding mentoring team and an exceptional training environment, I am ideally positioned to achieve the proposed scientific training and career development goals and mature into an independent investigator.