骨髓间充质干细胞源性外泌体microRNA拮抗矽肺纤维化作用及机制研究

Silicosis is one of the most serious occupational diseases in China and there is no effective treatment for it. Recent studies demonstrated that exosomes from mesenchymal stem cells played an important role in repairing tissue injuries. Our previous studies have found that epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of pulmonary fibrosis induced by silica and bone marrow mesenchymal stem cells (BMSCs) inhibit silicosis fibrosis due to paracrine action, suggesting that exosomes from BMSCs (BMSC-Exos) might be involved in the inhibition of EMT. However, the mechanisms underlying these actions remained elusive. Therefore, we suppose that miRNA in BMSC-Exos might inhibit EMT and pulmonary fibrosis induced by silica. In this research, technologies including Micro CT for lung tissue scanning, flexiVent Pulmonary Maneuvers, small animal in vivo imaging system and dynamic dust exposure for rats-silicosis model are applied to investigate the effects of BMSC-Exos on antagonizing EMT and pulmonary fibrosis induced by silica. In vitro, Three-Dimensional cell culture, small RNA sequencing and bioinformatics technology are used to screen out the key miRNA related to EMT in BMSC-Exos. Furthermore, lentivirus overexpression vector and siRNA interference technique are used to investigate the mechanism of key miRNA in BMSC-Exos antagonizing silica induced EMT. The research would provide innovative way for the further exploration of effective strategies on silicosis treatment.

矽肺是我国发病最多、健康危害最严重的职业病，尚无有效治疗方法。最新研究显示干细胞源性外泌体（Exos）具有组织再生潜能。课题组前期系列研究表明：上皮-间质转化（EMT）是石英致肺纤维化关键环节，骨髓间质干细胞（BMSCs）通过旁分泌作用拮抗矽肺纤维化，提示BMSCs源Exos（BMSC-Exos）可能参与抑制EMT过程，但机制不清。本课题设想BMSC-Exos中miRNA抑制石英诱导的EMT和肺纤维化。拟通过动态染尘制备大鼠矽肺模型，运用Micro CT、肺功能检测、小动物活体成像等技术，观察BMSC-Exos在石英致EMT及肺纤维化的作用；体外实验利用三维细胞培养、Small RNA测序和生物信息学方法筛选BMSC-Exos中与EMT相关的miRNA，应用慢病毒过表达载体及siRNA干扰技术，探讨Exos中关键miRNA拮抗石英诱导EMT机制。该研究将为探索矽肺新的治疗方法提供科学依据。

矽肺是我国发病最多、健康危害最严重的职业病，目前尚无有效的治疗方法。最新研究显示间充质干细胞源性外泌体（Exos）具有组织再生潜能。本研究采用3D floTrixTM动态培养系统培养人脐带间充质干细胞（hucMSCs），提取hucMSC-Exos；Small RNA测序和生物信息学方法筛选hucMSC-Exos中与EMT和成纤维细胞活化相关的miRNAs；应用慢病毒过表达载体及siRNA干扰技术，探讨hucMSC-Exos中关键miRNA拮抗石英诱导EMT和成纤维细胞活化的机制。体内实验通过非暴露式气管滴注法制备小鼠矽肺模型，运用Micro-CT、肺功能检测、小动物活体成像等技术，阐明hucMSC-Exos及hucMSC-Exos-miR-26a-5p(+)干预在石英致EMT及肺纤维化中的作用规律。研究发现：①通过高通量测序分析hucMSC-Exos中的差异miRNAs，发现与成纤维细胞活化密切相关的let-7i-5p及其靶基因TGFBR1，以及与EMT过程密切相关的miR-26a-5p及其靶基因Adam17；②HucMSC-Exos干预能够减轻石英导致的肺纤维化，并改善肺功能；③HucMSC-Exos中的let-7i-5p通过TGFBR1调控NIH-3T3细胞中p-Smad3的表达，抑制成纤维细胞的活化和细胞外基质的分泌，从而改善石英诱导的肺纤维化；④HucMSC-Exos通过递送miR-26a-5p至MLE-12细胞，抑制Adam17/Notch信号通路的激活，从而发挥拮抗TGF-β1诱导EMT的作用；⑤hucMSC-Exos-miR-26a-5p(+)干预能够拮抗石英诱导的肺EMT、抑制Adam17/Notch信号通路的激活，从而减轻石英导致的肺纤维化并改善肺功能。该项研究为寻找潜在的矽肺治疗有效途径提供了理论依据。