外泌体miRNA介导的细胞间通讯在实验性矽肺发生发展中的作用

The occurrence and development of silicosis is a regulatory process induced by silica dust via signaling transmission and interaction among a variety of cells, previous studies mainly focused on the intercellular communication among different corresponding cells induced by active molecules, but the mechanism and key targets have not yet been fully proved. The role of exosome in the intercellular communication has drawn more and more attention, which emerged an important role of exosomal miRNA in intercellular signaling transmission and regulation. This study was designed from the aspect of isolation and identification of exosomes ,which were excreted by cultured cells, to establish an experimental silicosis mice model, combined with population epidemiological survey, using fluorescent labeling, gene silencing, high-throughput chip sequencing, flow cytometry, transmission electron microscopy and other techniques to systematically study the biological characteristics of exosomal miRNAs of macrophages, dendritic cells, fibroblasts and T lymphocytes on the degree of participation, activity effects and regulatory pathways in the development of silicosis, and further to explore its impact on the mechanism of silicosis response, inflammatory response, gene expression, immune regulation, especially cell transdifferentiation, fibrosis and other processes, to deepen understanding the development mechanism of silicosis, to explore potential biomarkers and key targets for further monitoring and early detection of silicosis and provide new ideas and theoretical basis for the illustration of silicosis mechanisms.

矽肺发生发展是矽尘引起多种细胞藉由信号传递而相互作用的调控过程，以往主要研究活性分子为信号介导的矽肺效应细胞间通讯，但机制、关键靶点尚未彻底探明。外泌体在细胞间信号传递中的作用日益受到重视，推测矽肺过程中很可能存在外泌体miRNA介导的细胞间信号传递和调控效应。拟从细胞培养提取、鉴定外泌体入手，构建实验性矽肺小鼠模型，结合人群流行病学调查，采用荧光标记、基因沉默、高通量芯片测序、流式细胞术、透射电子显微镜等技术，系统研究巨噬细胞、树突状细胞、成纤维细胞及T淋巴细胞来源外泌体miRNA的生物学特性及其在矽肺发生发展中细胞间通讯的参与程度、活性效应、调控路径，深入探索其对机体二氧化硅粉尘应答、炎性反应、基因表达、免疫调节，尤其是细胞转分化、纤维化等过程的影响，深化对矽肺发生发展机制的认识，探讨潜在的生物标志和关键靶点，为进一步研制尘肺监测与早期发现的有效指标及干预措施提供新的思路和理论依据。

外泌体在细胞间信号传递中的作用日益受到重视。外泌体 mi RNAs 具有良好标识性、特异性和稳定性，但其在矽肺中的研究报道尚不多见。从细胞、动物、人群三个层面系统研究了外泌体miRNA生物学特性及其在矽肺发生发展中的作用与机制。分别采集54例尘肺患者、100例健康对照外周血标本及染尘小鼠巨噬细胞，外泌体基因测序发现尘肺病人、染尘巨噬细胞中miR-126a-5p、miR-335-5p、miR-122-5p、miR-125a-5p、miR-30c-2-3p和miR-107均高表达，而miR-27b-5p低表达。以矽肺病患者和健康对照各50例进行流行病学调查验证，病例组血清外泌体及其miR-27b-5p、miR-107、miR-122-5p、miR-125a-5p、miR-126-5p、miR-335-5p水平升高。体外实验发现SiO2粉尘可刺激巨噬细胞外泌体数量增多、构成改变，miR-107-3p可介导肺成纤维细胞转分化为肌成纤维细胞，其机制涉及TGF-β信号通路激活以及靶向抑制CDK6激活细胞分化。矽肺小鼠血清外泌体与肺组织中miR-107水平先降低后升高，miR-107抑制剂可减轻SiO2致肺纤维化作用；肺组织中miR-125a-5p水平明显升高，脾脏中Th1细胞比例减少，Th2、Th17细胞比例增多，纤维化水平升高，miR-125a-5p antagomir可明显抑制染尘小鼠肺组织纤维化。体外研究染尘巨噬细胞外泌体及其刺激的T细胞和成纤维细胞miR-125a-5p表达增高。另外，分离SiO2粉尘诱导的巨噬细胞外泌体进行蛋白质组学分析，发现与细胞增殖和分化高度相关的蛋白质SPP1在SiO2粉尘所致纤维化中发挥重要作用。研究扩展了对尘肺病发生发展机制的认识，发现外泌体具有潜在生物标志和靶标价值，为进一步深入研制尘肺病监测与早期发现指标及干预措施提供了新的思路和参考依据。