小气道和肺泡成纤维细胞在COPD基质转换中的区域性差异及其机制

Chronic obstructive pulmonary disease (COPD) yields huge disease burden in China. Available therapies cannot effectively slow disease progression. Pathological features of COPD are small airway remodeling and emphysema; the former is caused by excessive extracellular matrix (ECM) deposition or even fibrosis, and the latter is owe to insufficient ECM repair. It is unclear why adjacent tissue in the same lung present opposite ECM turnover. Fibroblasts are important effector cells to generate ECM and regulate its turnover, and preliminary research suggests that fibroblasts isolated from the peripheral lung tissue of patients with COPD showed impaired repair function and weakened response to TGF-β1. Thus we postulate regional differences in ECM turnover of fibroblasts between small airways and alveoli, and that TGF-β1 signaling might be the potential molecular mechanisms. In this project, we plan to use microdissection technique to isolate primary fibroblasts from small airways and alveoli tissue of patients with COPD and in rat COPD models for further culture and study. In vitro cell models are to be set up by fibroblast/airway epithelial cell co-culture and fibroblast/typeⅡalveoli epithelial cell co-culture. The heterogeneity of fibroblasts at different compartments are to be investigated into details from the following aspects: (1) cell migration, proliferation, and activation; (2) synthesis, deposition and degradation of ECM components; and (3) the responses to exogenous stimuli including TGF-β1, tobacco smoke, lipopolysaccharide, interleukin-6, interleukin-8, macrophage conditioned media, theophylline and corticosteroid. TGFβ1 signal transduction axis drift and the role of TGFβR3 are then to be investigated using PCR chip, protein phosphorylation antibody chips, dual luciferase reporter gene assays, siRNA and overexpression plasmid technology. The data from these studies will help to understand the development of COPD better and find new potential therapeutic targets.

慢性阻塞性肺疾病（COPD）是我国最大疾病负担之一，现有疗法不能有效延缓病情进展。COPD的病理特点是小气道壁重构和肺气肿，前者细胞外基质（ECM）沉积过多甚至纤维化，后者ECM修复不足，紧邻部位呈现相反的ECM转换，其成因不明。已知成纤维细胞是生成和调控ECM的重要细胞，我们的前期研究提示COPD外周肺组织成纤维细胞弹性纤维沉积障碍、对TGFβ1的反应异常，因此假设成纤维细胞在小气道壁和肺泡存在ECM转换的区域性差异，其机制在于TGFβ1信号转导通路。本项目拟用显微解剖技术分离COPD患者和大鼠小气道及肺泡成纤维细胞，建立体外细胞模型，从细胞迁徙、增殖、活化，ECM成分的合成、沉积、降解及对刺激的反应研究其区域性差异；用PCR芯片、蛋白磷酸化抗体芯片、双荧光素酶报告基因检测、siRNA及过表达质粒等技术研究TGFβ1信号转导的漂移及TGFβR3在其中的作用，为新的治疗靶点提供理论依据。

慢性阻塞性肺疾病（COPD，简称“慢阻肺”）的病理特点是小气道壁重构和肺气肿，前者表现为细胞外基质（ECM）在气道的沉积，而后者则是肺泡组织中ECM的减少、缺失。成纤维细胞作为肺组织内ECM的主要来源，我们之前的研究已经证实慢阻肺患者肺泡组织的原代成纤维细胞的增殖能力显著低于正常对照组。然而，慢阻肺气道和肺泡这两个不同部位的成纤维细胞表型是否具有差异未被阐明。在本研究中，我们在复制慢阻肺小鼠模型的基础上，采用显微解剖技术分离、培养小鼠气道和肺泡部位的原代成纤维细胞，采用western blot、RT-qPCR以及Alamar Blue方法较为全面地评价了气道和肺泡部位成纤维细胞在ECM的产生和增殖能力方面的差异性，以及介导气道和肺泡成纤维细胞产生不同ECM的机制。研究结果表明，慢阻肺小鼠气道部位成纤维细胞的ECM的表达量明显高于肺泡部位成纤维细胞ECM的表达量，同时慢阻肺小鼠气道部位成纤维细胞的增殖能力显著高于肺泡部位成纤维细胞；其次，慢阻肺气道和肺泡部位原代成纤维对外源性TGF-β1的反应具有显著差异性：与刺激前相比，气道部位成纤维细胞产生的ECM明显增多，而肺泡部位成纤维细胞ECM的表达量无明显差异；同时，经过刺激的气道部位成纤维细胞的增殖能力显著增加，而肺泡部位成纤维细胞基本无变化。最后，不同与以往的研究，我们发现在慢阻肺气道和肺泡ECM的产生过程中，不仅TGF-β1/Smad2/3通路起作用，TGF-β1/Smad1通路在区域性差异中可能起着重要的作用。综上，慢阻肺小鼠气道和肺泡部位成纤维细胞表型存在明显差异，主要表现为ECM的改变、增殖能力以及对TGF-β1反应性的不同，而且TGF-β1/Smad1通路可能参与了气道和肺泡成纤维细胞的区域性差异。此外，通过基因芯片、数据挖掘以及生物信息学方法筛选了肺气肿的可能致病基因和干预靶点；通过特异性敲低的方法研究Versican的作用及作为干预靶点的可能性。

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