肺泡巨噬细胞源性细胞外泌体调控硅肺肌成纤维细胞表型转化机制研究

Silicosis, the fibrotic lung disease, greatly threatens human health. The regulation mechanism on phenotype transition of myofibroblasts in fibrotic tissues remained to be determined. We previously found that the supernatant of silica-stimulated alveolar macrophages (AM) could induce phenotype transition of fibroblasts into myofibroblasts. Exosomes, duing to the comunication between cells, cause researchers great interest. We also detected the presence of exosomes in the supernatant of silica-stimulated AM. Whether AM-derived exosomes affect phenotype transition of myofibroblasts and fibrotic progression in silicosis is unclear. Based on our findings, the present study is to clarify the regulation mechanism on phenotype transition of myofibroblasts in silicosis. In vitro, we isolate exosomes and identify differentially express molecules in supernatant of silica-stimulated rat AM and take exosomes-incubated rat lung fibroblasts as a model. Furthermore, the endoplasmic reticulum (ER) stress is investigated, and prevention methods are performed to evaluate the roles of AM-derived exosomes, ER stress in myofibroblasts phenotype transition. In vivo, we isolate AM-derived exosomes and verify the differentially express molecules in silicotic rat and patients, determining the relationship between AM-derived exosomes and ER stress, myofibroblasts phenotype transition, fibrotic progression. The prospective resulsts may provide scientific evidences for prevention and treatment of silicosis.

硅肺纤维化危害非常严重。纤维化局部肌成纤维细胞表型转化调控机制一直是纤维化研究函待解决的问题。我们前期发现二氧化硅刺激的肺泡巨噬细胞(AM)上清液可以诱导成纤维细胞表型转化为肌成纤维细胞。细胞外泌体因其在细胞之间通讯的功能,而引起学者们极大的兴趣。我们同时检测到二氧化硅刺激的AM上清液中细胞外泌体的存在，AM是否通过细胞外泌体影响肌成纤维细胞表型转化，从而促进硅肺纤维化进展？本项目通过体外、体内两个层面的研究，探讨肺肌成纤维细胞表型转化调控机制：体外提取二氧化硅刺激的大鼠AM上清液中的细胞外泌体并鉴定差异表达分子;以细胞外泌体与大鼠肺成纤维细胞共孵育为模型，检测内质网应激分子活化情况，再通过干预实验明确调控机制。体内提取硅肺病人、大鼠硅肺模型肺泡巨噬细胞源性的细胞外泌体并验证差异表达分子，研究其与内质网应激、肌成纤维细胞表型转化、纤维化进展的关系。这将为硅肺的防治提供科学实验依据。

硅肺病是传统的职业病且预后不佳，是由于长时间吸入游离二氧化硅粉尘（SiO2）引起的慢性纤维化肺疾病。肺泡巨噬细胞是硅肺进展中的关键效应细胞，而外泌体是细胞间通讯的重要媒介，这表明肺泡巨噬细胞来源的外泌体在硅肺发病机制中具有潜在的作用。本课题主要在体内和体外实验中探讨肺泡巨噬细胞来源的外泌体是否促进或抑制硅肺纤维化进展，并探讨其作用机制。在体外实验中，我们证实了二氧化硅暴露的巨噬细胞分泌的外泌体（SiO2-Exos）含量增加；SiO2-Exos促进肌成纤维细胞表型转化，增殖和迁移，这些作用依赖于内质网应激相关信号通路的激活。通过miRNA高通量测序检测SiO2-Exos中差异表达的miRNA分子，我们筛选出148个差异表达的外泌体miRNAs。与NC-Exos组相比，SiO2-Exos组中miR-365-3p和miR-7219-3p中表达显著上调，而miR-378d呈显著下调。SiO2-Exos组中上调的miR-365-3p可能通过靶向Pten调控肌成纤维细胞表型转化。在体内实验中，我们发现，相较于生理盐水对照组，硅肺模型组小鼠的肺泡灌洗液中外泌体分泌数目显著增加，这些外泌体主要来源于肺泡巨噬细胞。外泌体分泌抑制剂GW4869可减轻二氧化硅暴露导致的肺组织炎症及纤维化程度。miRNA高通量测序结果显示，相较于对照组，硅肺组肺泡灌洗液来源的外泌体中129个miRNA表达上调，212个miRNA表达下调；血清来源的外泌体中74个miRNA表达上调，9个miRNA表达下调。RT-PCR结果显示硅肺组肺泡灌洗液来源的外泌体中miR-125a-5p、miR-125b-5p及miR-199b-5p表达下调，miR-7b-5p表达上调；血清来源的外泌体中miR-24-3p表达上调，miR-214-3p表达下调。与对照组相比，miR-365-3p、miR-7219-3p在硅肺小鼠肺组织中表达上调，而miR-378d、miR-125a-5p及miR-214-3p表达下调。其中，下调的肺泡巨噬细胞来源的外泌体miR-125a-5p通过靶向调节STAT3表达水平调控肌成纤维细胞表型转化。总之，本课题研究发现暴露于二氧化硅的肺泡巨噬细胞其外泌体分泌增加，外泌体通过携带特异性的miRNA分子调控硅肺纤维化进展，这将为硅肺纤维化防治提供新的思路。