外泌体microRNAs介导碳纳米管暴露所致肺纤维化的作用及机制研究

The environmental and health risks of carbon nanotubes (CNTs) are currently of great interest for a variety of production and industrial applications. Several studies have showed that certain forms of CNTs are potent fibrogenic inducers animal pulmonary fibrosis (PF), and the exosomes and microRNAs (miRNAs) play an important role in many chronic diseases such as fibrosis process. However, the function and mechanism of exosomes and its miRNAs on PF induced by CNTs is still unknown. In the present study, we will re-established a mouse model of CNTs-induced pulmonary fibrosis. Meanwhile, the exosomes secretion of CNTs-injured macrophages and alveolar epithelial cells will be investigated, to verify weather the exosomes play a regulatory role in fibroblast activation, and ultimately differentiate into myofibroblast on cellular levels. In oder to identify the potential roles of miRNAs by regulating targeted fibrotic signaling pathways genes, the effect and mechanism of CNTs-induced pulmonary fibrosis will be systematically studied through exosomes miRNA microarray, screening ectopic miRNAs related PF, intervening the miRNAs expression by mimics/inhibitors in fibroblast and validating by RT-qPCR, Western-Blot and FISH. This proposal will make clear the function of exosome and its miRNAs in the CNTs-induced PF at animal, cellular and molecular levels, and will provide experimental evidence and theoretical basis for environmental risk assessment of nanmaterials.

碳纳米管(CNTs)的环境健康风险因其大量生产和应用而受到广泛关注。多项研究发现CNTs具有致动物肺纤维化(PF)，外泌体及其miRNAs在纤维化等慢性疾病进展中起重要作用。但外泌体及miRNAs在CNTs导致PF过程中的作用和机制仍不清楚。我们拟建立CNTs诱导小鼠PF模型，在细胞水平构建CNTs暴露巨噬细胞和上皮细胞外泌体诱导成纤维细胞活化模型，明确外泌体在CNT致PF中的作用。通过外泌体miRNA芯片，分析CNTs暴露诱导的miRNAs表达变化，筛检与PF相关差异miRNAs，miRNA模拟物或siRNA技术干预，RT-qPCR、Western-Blot和原位免疫染色等方法验证，明确miRNA对纤维化信号通路的调节作用。本项目在动物、细胞和分子水平上阐明外泌体及其miRNAs在CNTs致PF的作用和分子机制，为全面评价纳米材料的健康风险提供一定的科学线索和依据。

碳纳米管(CNTs)是一种大量生产和在生物医学、环境处理和电子传感等领域广泛应用的纳米材料，在环境介质中也广泛存在，因此研究其环境健康效应具有重要意义。前期研究发现CNTs具有致动物肺损伤的风险，但外泌体及其组份在CNTs导致肺纤维化过程中的作用和机制仍不清楚。本项目通过首先通过药物博来霉素诱导小鼠肺纤维化模型，确认肺纤维化模型的病理和分子特征。后通过呼吸暴露MWCNTs小鼠，结果表明MWCNTs暴露小鼠表现出肺纤维化特征，肺泡塌陷、破坏、肺实质增加，暴露组血清中 TGF-β1 的含量明显升高。项目还对CNTs暴露巨噬细胞RAW264.7后其分泌外泌体的变化进行探讨，发现CNTs的暴露不改变巨噬细胞分泌外泌体的大小，但会增加其分泌外泌体的蛋白含量。CNTs呼吸暴露小鼠后，肺泡灌洗液中分泌的外泌体形貌和大小没有发生改变，5d暴露增加外泌体中蛋白的含量。因此，CNTs的呼吸暴露有导致肺纤维化的健康风险，且会影响细胞以及机体分泌外泌体的组份。