Genomic Analysis of Tissue and Cellular Heterogeneity in IPF

 描述(申请人提供)：特发性肺纤维化(IPF)是一种慢性进行性肺部疾病，具有显著的发病率和死亡率。目前还没有有效的 肺移植以外的治疗。IPF肺表现出不同的mRNA和microRNA表达模式以及DNA甲基化的全局变化。最近的观察，主要是在肺纤维化的动物模型中，表明异常在多个途径中起作用，如凝血、细胞凋亡、氧化应激、上皮细胞表型转变、内质网应激和发育途径。支持这项研究的假设是，IPF独特的组织病理学特征--时间上的异质性、肺泡细胞增生、丰富的肌成纤维细胞灶和异常重构--代表了IPF特有的分子疾病机制。因此，了解这些特征背后的分子网络将有助于更好地理解IPF，并最终更合理地进行基于疾病机制的治疗干预。为此，我们组建了一支由肺纤维化、基因组学、计算生物学、计算机科学、细胞和分子生物学、统计学、高通量筛查和生物信息学等多学科专家组成的团队。这项研究将承担以下具体目标：1)鉴定组织学定义的肺微环境的独特基因组和转录组特征。这一目标将包括使用microCT引导的显微解剖、下一代测序和激光捕获显微解剖-简化代表亚硫酸氢盐测序(LCM-RRBS)来生成组织学上不同的、不同的受影响区域的mRNA、microRNA和表观基因组图谱。2)通过对IPF肺中不同细胞群(肌纤维母细胞、增生性上皮细胞)的LCM引导采样、从患者分离的原代细胞(肺泡II型和成纤维细胞)的转录转录图谱的组合，确定IPF肺中细胞对基因组和表观基因组变化的贡献 在基线有和没有IPF的情况下，以及对纤维化相关扰动的反应。细胞签名将通过定量免疫组织化学和原位杂交在IPF肺中进行验证、定位和量化。3)基于基因组数据建立IPF的动态调控模型，并对模型预测进行初步的实验验证。这一目标包括生成综合的IPF基因组和表观基因组数据纲要，应用新的分析方法来确定关键调控因素，并通过测试潜在数据和 分析将被合并到一个简单、直观、基于网络的界面IPFmap中，该界面将允许研究人员以交互方式挖掘数据，使用分析工具，将他们自己的数据整合到这些分析中，并提供对辅助数据库的无缝访问，从而支持治疗的开发。

 DESCRIPTION (provided by applicant): Idiopathic Pulmonary Fibrosis (IPF) is a chronic and progressive lung disease with significant morbidity and mortality. At present there is no effective treatment other than lung transplantation. The IPF lung displays distinct patterns of mRNA and microRNA expression patterns and global changes in DNA methylation. Recent observations, mainly in animal models of lung fibrosis, suggest a role for aberrations in multiple pathways such as coagulation, apoptosis, oxidative stress, shifts in epithelial cell phenotypes, endoplasmic reticulum stress, and developmental pathways. The hypothesis underlying this grant is that the unique histopathologic features of IPF-temporal heterogeneity, alveolar cell hyperplasia, abundance of myofibroblast foci and aberrant remodeling-represent a molecular disease mechanism specific to IPF. Therefore, understanding the molecular networks that underlie these characteristics will lead to better understanding of IPF and eventually more rational, disease mechanism based therapeutic interventions. For this purpose we have assembled a multi-disciplinary team of experts in lung fibrosis, genomics, computational biology, computer science, cell and molecular biology, statistics, high-throughput screening and bioinformatics. The study will undertake the following specific aims: 1) Identification of the unique genomic and transcriptomics characteristics of histologically defined lung microenvironments. This aim will include generation of mRNA, microRNA and epigenomic profiles of histologically distinct, differentially affected regions of the lung using microCT guidd microdissection, next generation sequencing and laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS). 2) Determination of the cellular contribution to the genomic and epigenomic changes in the IPF lung by a combination of LCM guided sampling of distinct cell populations in the IPF lung (myofibroblasts, hyperplastic epithelial cells), transcriptomic profiling of primary cells (alveolar type II and fibroblasts) isolated from patients with and without IPF at baseline and in response to fibrosis relevant perturbations. Cellular signatures will be validated, localized and quantified in IPF lungs by quantitative immunohistochemistry and in-situ hybridization. 3) Generate a dynamic regulatory model of IPF based on genomic data and perform preliminary experimental validation of model predictions. This aim includes generation of an integrated IPF genomic and epigenomic data compendium, application of novel analytic approaches to identify key regulators and performance of preliminary validation of predictions by testing effect of perturbations of potential The data and analyses will be incorporated into a simple, intuitive, web-based interface, IPFmap, that will allow investigators to interactively mine the data, use analytical tools, integrate their own data into these analyses, and provide seamless access to complementary databases enabling development of therapies.