The role of Pdgfra+ fibroblasts in lung fibrosis and alveolar homeostasis

项目摘要/摘要 职业发展目标/计划：这个项目的目标是提供支持和持续的指导 发展成为研究肺纤维化和上皮细胞作用的独立内科科学家- 间充质相互作用在疾病发病机制中的作用。该课程是专门为 加强我对小鼠肺纤维化模型的理论和实践知识基础 表型分析和人类肺细胞生物学。这将通过动手实验室来实现。 经验、教学培训、数据和结果的介绍、与相关领域专家的互动以及 由一个专门的咨询委员会提供指导。正如这份提案所概述的，我将至少有75%的时间 为了研究而受到保护。在此期间，我将能够在许多新的领域获得实用的专业知识 技术，包括流式细胞术，荧光激活细胞分选(FACS)，评估小鼠 呼吸力学，以及人肺细胞分离和培养。我还将建立我的知识库 基因组学和基因表达分析的相关领域。我做得很好，可以成功地完成 鉴于我的经验丰富的导师和丰富而充满活力的研究环境，提案中概述的工作 我将执行这项工作。 研究项目：特发性肺纤维化(IPF)是一种以严重和进行性为特征的疾病 肺泡纤维化，最常发生在老年人身上，除肺外没有有效的治疗方法 移植。基于人类基因研究揭示了2型特异性基因突变之间的联系 肺泡上皮细胞(AEC2)与家族性肺纤维化 肺泡上皮细胞与疾病的发展有关。然而，我们缺乏关于这些是如何 上皮异常触发潜在间质的旺盛反应，从而导致 显著的进行性疤痕形成。我们也不知道是哪些细胞引起了 肌成纤维细胞是这种疾病的特征。我已经确定了一组独特的成纤维细胞 肺间充质，可能是肌成纤维细胞的来源。这些表达PDGF受体的细胞 α(PDGFRA)，靠近AEC2，在博莱霉素肺损伤后，增殖，上调 肌成纤维细胞标志物，并表达促纤维化介质，如透明质酸合成酶2(Has2)。我有过 还表明这些细胞支持小鼠AEC2的克隆生长和分化 小鼠培养系统，提示它们是肺泡上皮细胞生态位的重要组成部分。我 假设PDGFRA+成纤维细胞是博莱霉素肺损伤后肌成纤维细胞的来源，这些 当被触发时，细胞会产生促纤维化介质，如透明质酸(HA)和Has2，这些细胞 是人类AEC2生态位在动态平衡中的关键组成部分。 我将在以下具体目标中检验这些假设：目标1：确定PDGFRA+成纤维细胞是否 博莱霉素肺损伤后向肌成纤维细胞转化。目的2：验证PDGFRA+成纤维细胞是一种 促纤维化介质HA和Has2在博莱霉素性肺损伤中的主要来源 纤维化症。目的3：验证原代人PDGFRA+成纤维细胞支持存活的假设， 人类AEC2的增殖和分化，从而成为AEC2生态位的组成部分。

PROJECT SUMMARY/ABSTRACT Career development Goal / Plan: The goal of this project is to provide support and continued mentorship as I develop into an independent physician-scientist studying pulmonary fibrosis and the role of epithelial- mesenchymal interaction in the pathogenesis of the disease. The curriculum is specifically designed to strengthen my theoretical and practical knowledge base in murine models of lung fibrosis, genetic and phenotypic analysis, and human lung cell biology. This will be achieved through hands on laboratory experience, didactic training, presentation of data and results, interactions with experts in related fields, and mentorship from a dedicated advisory committee. As outlined in this proposal, I will have at least 75% of time protected for research. During this time, I will be able to gain practical expertise in a number of new techniques, including flow cytometry, fluorescence activated cell sorting (FACS), assessment of murine respiratory mechanics, and human lung cell isolation and culture. I will also build my knowledge base in relevant areas of genomics and gene expression analysis. I am well positioned to successfully complete the work outlined in the proposal given my experienced mentors and the rich and dynamic research environment in which I will perform the work. Research Project: Idiopathic pulmonary fibrosis (IPF) is a disease characterized by severe and progressive alveolar fibrosis that occurs most often in older individuals and that has no effective treatment short of lung transplant. Based upon human genetic studies revealing a link between mutations in genes specific to type 2 alveolar epithelial cells (AEC2s) and familial forms of pulmonary fibrosis, it is accepted that abnormalities in the alveolar epithelium are associated with disease development. We lack knowledge, however, of how these epithelial abnormalities trigger the exuberant response from the underlying mesenchyme that leads to significant and progressive scar formation. We also are unaware of which cells give rise to the pathologic myofibroblasts that are characteristic of the disease. I have identified a unique population of fibroblasts in the lung mesenchyme that are likely to be a source of myofibroblasts. These cells, which express PDGF receptor alpha (Pdgfra), lie in close proximity to AEC2s and, after bleomycin lung injury, proliferate, up-regulate myofibroblast markers, and express pro-fibrotic mediators such as hyaluronic acid synthase 2 (Has2). I have also shown that these cells support the clonal growth and differentiation of murine AEC2s in a 3-dimensional murine culture system, suggesting that they are critical components of the alveolar epithelial cell niche. I hypothesize that Pdgfra+ fibroblasts are a source of myofibroblasts following bleomycin lung injury, that these cells produce pro-fibrotic mediators such as hyaluronan (HA) and Has2 when triggered, and that these cells are critical components of the human AEC2 niche during homeostasis. I will test these hypotheses in the following specific aims: AIM 1: To determine if Pdgfra+ fibroblasts give rise to myofibroblasts after bleomycin lung injury. AIM 2: To test the hypothesis that Pdgfra+ fibroblasts are a major source of the pro-fibrotic mediators HA and Has2 in the development of bleomycin-induced pulmonary fibrosis. AIM 3: To test the hypothesis that primary human PDGFRa+ fibroblasts support the survival, proliferation, and differentiation of human AEC2s, thereby serving as components of the AEC2 niche.