Analysis of Alveolar Type II cells in Normal and Fibrotic Human Lung

描述(申请人提供)：特发性肺纤维化(IPF)占间质性肺疾病病例的一半以上。患有IPF的患者发展为进行性肺功能丧失，平均在确诊后3年死亡。除纤维化外，肺泡II型细胞在IPF肺中也有异常。然而，它们在疾病发病机制中的作用仍不清楚。这一建议检验了一种新的假设，即上皮细胞通过转变为分泌胶原的间充质细胞直接参与肺纤维化的形成。在IPF肺内，有一群易于转化为成纤维细胞的上皮细胞。这些上皮源性成纤维细胞是特发性肺纤维化发生的基质重塑和纤维化的主要来源。解决这一假设的具体目标是：1)建立从IPF患者肺组织中纯化的肺泡II型细胞的独特表型特征。2)分离和鉴定IPF肺组织中表达CD15的肺泡II型细胞亚型。方法：为了达到这些目的，我们将利用流式细胞仪对E-钙粘素进行分选，从正常肺和特发性肺纤维化肺中分离纯化的II型细胞群。基因表达阵列、RT-PCR和免疫组织化学将用于确定IPF II型细胞特有的基因表达变化，这些变化可能有助于疾病的发病机制。促进IPF II型细胞上皮向间充质转化(EMT)的候选基因的作用将在EMT的体外试验中得到证实。在目标2中，将使用FACS分选来分离表达细胞表面标记CD15的II型细胞亚型。这一亚型占IPF肺中II型细胞的6%。纯化的CD15+型细胞分离株将与CD15-II型细胞进行比较，以了解它们的增殖、凋亡或向间充质细胞转化的能力。基因表达阵列将被用来定义它们独特的遗传表型及其与IPF发病机制的关系。意义：通过完成建议的目标，将解决在IPF发病机制和基础肺生物学方面的重大知识空白，包括：1)这些研究将提供新的数据，特别是正常和纤维化II型细胞之间的表型差异。2)他们将确定EMT发生在IPF肺和这一过程的介体中；3)他们将首次建立存在于人肺内的特定的II型细胞亚型和这种表型的独特表型特征；4)除了促进我们对肺纤维化发病机制的理解外，通过这些实验开发的新数据和技术还可以用于未来对IPF发病机制和人类肺泡II型细胞生物学的研究。 公共卫生相关性：特发性肺纤维化是肺纤维化最常见的形式，是一种平均存活三年的致命疾病。这项建议应用新技术来纯化和鉴定特发性肺纤维化患者肺组织中的肺泡II型细胞和II型细胞亚型(CD15+)。在纯化后，将建立IPF II型细胞和CD15+II型细胞独特的生物学特性。通过广泛分析IPF II型细胞的独特生物学，这些研究有可能确定特发性肺纤维化的意外原因，并可能导致新疗法的开发，有利于调节这种毁灭性疾病的结果。

DESCRIPTION (provided by applicant): Idiopathic pulmonary fibrosis (IPF) accounts for more than half of the cases of interstitial lung disease. Patients suffering from IPF develop a progressive loss of lung function and die, on average, 3 years after diagnosis. In addition to fibrosis, alveolar type II cells are abnormal in IPF lungs. However, their role in disease pathogenesis remains undefined. This proposal examines the novel hypothesis that epithelial cells directly participate in the formation of lung fibrosis by transitioning into collagen secreting mesenchymal cells. Within IPF lung there is a subpopulation of epithelial cells predisposed to transition into fibroblasts. These epithelial derived fibroblasts are a major source of the matrix remodeling and fibrosis that occurs in IPF lung. Specific aims pursued to address this hypothesis are: 1) To establish the unique phenotypic characteristics of alveolar type II cells purified from lungs of IPF patients. 2) To isolate and characterize an alveolar Type II cell subtype expressing CD15 found in IPF lungs. Approach: To pursue these aims, pure populations of Type II cells will be isolated from normal and IPF lungs using FACS sorting for E-cadherin. Gene expression arrays, rt-PCR, and immunohistochemistry will be used to define changes in gene expression unique to IPF Type II cells that may contribute to disease pathogenesis. The role of candidate genes that promote epithelial to mesenchymal transition (EMT) of IPF Type II cells will be confirmed in an in vitro assay of EMT. In Aim 2, FACS sorting will be used to isolate a Type II cell subtype expressing the cell surface marker CD15. This subtype represents up to 6% of Type II cells in IPF lungs. Purified isolates of CD15+ type cells will be compared to CD15- Type II cells for their ability to proliferate, undergo apoptosis, or transition to mesenchymal cells. Gene expression arrays will be used to define their unique genetic phenotype and how it relates to the pathogenesis of IPF. Significance: By completing the proposed aims, significant gaps in knowledge of the pathogenesis of IPF and basic lung biology will be resolved including; 1) The studies will provide new data that specifically phenotype differences between normal and fibrotic Type II cells. 2) They will establish that EMT occurs in IPF lungs and the mediators of this process, 3) They will establish for the first time a specific Type II cell subtype that exists within human lung and the distinct phenotypic characteristics of this phenotype, 4) In addition to advancing our understanding of the pathogenesis of lung fibrosis, the new data and technologies developed via these experiments can be used in future studies of the pathogenesis of IPF and the biology of alveolar Type II cells in human lung. PUBLIC HEALTH RELEVANCE: Idiopathic pulmonary fibrosis, the most common form of lung fibrosis, is a deadly disease with average survival of three years. This proposal applies novel technologies to purify and characterize alveolar Type II cells and Type II cell subtypes (CD15+) from the lungs of patients with idiopathic pulmonary fibrosis. Following purification, the unique biological characteristics of IPF Type II cells and CD15+ Type II cells will be established. By broadly analyzing the unique biology of IPF Type II cells, these studies have the potential to identify unsuspected causes of Idiopathic pulmonary fibrosis and could lead to the development of new therapies that favorably modulate outcomes of this devastating disease.