外泌体miR-21经PTEN/PI3K/VEGF途径介导哮喘气道上皮屏障损伤的机制研究

The airway epithelial barrier plays a crucial role in activation of asthma airway inflammation. Airway epithelium-derived exosomes involved in mediating asthmatic airway inflammation, but its effect on airway epithelial barrier function is unclear. In our previous study, we’ve demonstrated that common allergens house dust mite(HDM) can disrupt airway barrier by PTEN/PI3K/VEGF pathway. Further, human bronchial epithelial cells (HBECs) exposed to HDM significantly increase the release of exosomes.These exosomes increased FITC-dextran permeability of epithelial layer,down-regulated PTEN and increased VEGF expression. Exosomal miRNA microarray analysis revealed that miR-21 increased in HDM-induced HBEC-derived exosomes.Luciferase report showed miR-21 inhibit PTEN expression.This indicates that exosomes can damage airway epithelial barrier function, and its mechanism may be related to its content miR-21 regulation of PTEN/PI3K/VEGF axis.. This study will isolate airway epithelium-derived exosomes from asthma model by using ultrafiltration centrifugalization. Then we investigate the effects of exosomes derived from HDM–treated on epithelial barrier function and pathway protein PTEN，PI3K/Akt and VEGF expression. Meanwhile we use miR-21 mimics and miR-21 inhibitors to deep explore the role and the possible mechanism of exosomal miR-21 on asthmatic airway epithelial barrier dysfunction via PTEN/PI3K/VEGF pathway both in vitro and in vivo. The project aims to find new targets for therapeutic intervention in airway epithelial injury and provide new insights in the pathogenesis of asthma.

气道上皮屏障损伤是哮喘启动的关键环节，上皮来源的外泌体参与介导哮喘气道炎症，但其对气道上皮屏障功能的影响目前尚不明确。我们研究显示，PTEN/PI3K/VEGF途径参与哮喘致敏物HDM所致的气道上皮屏障损伤。HDM刺激气道上皮外泌体释放，这些外泌体可降低气道上皮细胞PTEN表达，上调VEGF并破坏屏障功能。且外泌体miR-21在HDM刺激的气道上皮细胞富集，miR-21可抑制PTEN mRNA表达。提示外泌体介导气道上皮屏障损伤，机制与其内容物miR-21调控PTEN/PI3K/VEGF轴相关。本项目拟利用超速离心技术提取哮喘模型上皮源性外泌体，探讨其对上皮屏障功能及通路蛋白PTEN、PI3K和VEGF的影响。同时拮抗miR-21，在体内外研究外泌体miR-21经PTEN/PI3K/VEGF轴调控哮喘气道上皮屏障损伤的机制，为寻找哮喘气道上皮屏障受损的新靶点，完善哮喘发病机制提供理论基础。

支气管哮喘是一种涉及基因和环境相互作用的慢性气道炎症性疾病，全球哮喘患病率呈逐年上升的趋势，而目前哮喘发病机制的未完全明了，部分重症难治性哮喘患者对糖皮质激素反应不佳，这均为哮喘的预防和治疗带来了一定困难。气道上皮细胞损伤在哮喘气道炎症启动和维持中发挥着关键作用，进一步阐明哮喘气道上皮屏障功能受损的关键机制及寻找控制哮喘气道炎症的新靶点是重要的研究目标。本研究中，我们采用FITC-透过率验证外泌体对气道上皮屏障的影响。电镜、粒径测定和Western blot用于检测外泌体样本，用qPCR、纳米流式检测外泌体miR-21表达及分选，并使用囊泡抑制剂研究外泌体在此过程中所起作用。Mimics瞬时转染、Western blot等检测外泌体miR-21对PTEN的影响。.本项目获得以下成果：1.HDM可以促进气道上皮细胞外泌体miR-21上调及分选，抑制外泌体分泌可导致miR-21表达下调。进一步实验证实，HDM外泌体及miR-21 mimics可抑制PTEN的表达，HDM外泌体可升高气道上皮细胞FITC-右旋糖酐通透率。综上我们可以证明，外泌体miR-21通过调节PTEN，参与调控哮喘气道上皮屏障损伤。2.我们建立了OVA/CFA/LPS诱导的小鼠模型可以很好地模拟人难治性哮喘气道的复杂变化，证实在CFA诱导小鼠模型中性粒细胞炎症更明显，由此确定了一种新的中性粒细胞哮喘小鼠模型。本研究为寻找干预哮喘气道上皮屏障受损的新靶点，完善哮喘发病机制提供理论基础。