外泌体miR-21调控气道上皮细胞与巨噬细胞交互对话对COPD小气道重塑的影响及机制研究

Small airway remodeling is an indispensable pathological foundation of airflow limitation caused by chronic obstructive pulmonary disease(COPD). Research have found that epithelial-mesenchymal transition(EMT) might play as an important role in the process of small airway remodeling. The exosome is considered as a key medium in cell-to-cell biochemical information transmission,but it is uncertain whether exosome contribute to the regulation of EMT occurred in COPD or not. Our previous research has proved that the serum free miR-21 was increased in COPD patients which mainly derived from exosome. Meanwhile, our previous study has also found an elevated expression of miR-21 in airway epithelial cells and macrophages under the interference of cigarette smoke extract(CSE) which could mediate the polarization of macrophages toward the M2 phenotype. The M2 macrophage might induce EMT by secreting TGF-β . However, whether miR-21 secreted from exosome and participated in the interaction between airway epithelial cells and macrophages or not, is yet to be confirmed. This research is planning to observe the status of EMT in COPD patients, animal COPD models and relative cells, attempting to certify CSE is able to induce airway epithelial cells to produce exosome-contained miR-21, which could subsequently induce macrophages polarize toward the M2 phenotype, influencing the EMT status of the epithelial cells by regulating the Smad/TGFβ pathway, thereby participating the process of small airway remodeling, which may represent a new intervention target in the treatment of COPD.

小气道重塑是引起COPD气流受限重要的病理基础，研究发现上皮细胞间质转化（EMT）可能在其中扮演重要角色。外泌体（exosome）被认为是细胞间交流的关键介质，但exosome是否参与调控COPD的EMT尚不明确。我们前期研究证实COPD患者外周血清游离miR-21表达增加，而游离miRNA主要来源于外泌体，同时miR-21在CSE干预的上皮细胞及巨噬细胞中表达均增加，其可介导巨噬细胞向M2分化，M2可能通过分泌TGFβ参与EMT。但exosome miR-21是否参与气道上皮细胞与巨噬细胞的交互对话进而影响EMT有待进一步求证。本研究拟从COPD患者、动物模型和细胞水平观察COPD是否存在EMT，并证实CSE引起上皮细胞产生exosome miR-21进而诱导巨噬细胞朝M2分化，M2通过Smad/TGFβ通路影响上皮细胞EMT，从而参与小气道重塑过程，为COPD的治疗探索新的途径。

慢性阻塞性肺疾病（COPD）是目前全球范围内发病率及死亡率最高的疾病之一，严重危害人类健康。小气道重塑是其重要病理基础，上皮细胞间质转化（EMT）可能在其中扮演重要角色。但目前尚无针对气道重塑药物。我们前期研究发现，COPD患者外周血游离的miRNA-21表达增加,而血清miRNA-21主要来源于外泌体。miRNA-21可以介导巨噬细胞的M2定向极化，促进TGF-β分泌从而参与上皮间质化。本项目旨在阐明外泌体miRNA-21参与巨噬细胞与支气管上皮细胞之间的交互对话及其分子机制。. 本项目主要研究内容包括从COPD患者、动物模型和细胞水平观察COPD是否存在EMT，确定miR-21在COPD小气道重塑的EMT过程中的作用，研究外泌体 miR-21参与肺泡上皮细胞与巨噬细胞交互对话及对EMT影响的分子机制，为COPD的治疗探索新的途径。. 我们在本项目研究中发现COPD模型小鼠的肺支气管上皮存在EMT，这一现象在miRNA-21基因敲除后可以被部分抑制。体外实验表明香烟烟雾提取物以及TGF-β1激动剂均可引起支气管上皮细胞的EMT,且其程度与TGF-β1激动剂浓度密切相关。我们发现高剂量的CSE虽会上调支气管上皮细胞外泌体miRNA-21的含量，但也会引起支气管上皮细胞的过度凋亡，而低剂量的香烟烟雾提取物刺激支气管上皮细胞可引起其外泌体miRNA-21含量减少，与巨噬细胞共培养以后可抑制巨噬细胞的M2极化，并降低培养上清中的TGF-β1的水平，抑制EMT，这可能揭示了外泌体在COPD中的一种新的代偿作用。巨噬细胞M2极化程度与其所内化的外泌体miR-21含量呈正相关；miR-21模拟剂/抑制剂可以促进/阻断CSE暴露后支气管上皮细胞的自噬以及凋亡，可以放大/部分阻断巨噬细胞M2极化；另外，对支气管上皮细胞进行Smad7过表达达/Smad7siRNA敲低，其EMT水平分别被下调或者上调。说明SMAD7/TGF-β1通路可能介导了CSE诱导的支气管上皮细胞EMT过程。. 本项目研究表明低剂量CSE刺激支气管上皮细胞后可能通过降低外泌体miR-21水平减轻巨噬细胞M2极化，M2巨噬细胞通过Smad/TGF-β通路影响上皮细胞EMT，进而影响COPD小气道重塑，参与COPD发生。为COPD的治疗提供了新的理论基础和干预靶点。