基于外泌体介导的碳纳米材料炎性反应及分子机制研究

The research on the safety of nanomaterials is lagging far behind the rapid development of nanotechnology, which restricts the application of nanomaterials. Therefore, it is necessary to explore the safety of nanomaterials. Recently, the wide use of carbon nanomaterials (CNMs) has attracted a great deal of concerns on their potential adverse effects on human health, especially on the inflammatory effect. Lately, exosomes, function as important mediator in cell-cell communication, have been validated to mediate the inflammatory effect through releasing inflammatory molecules to their targets. However, the molecular mechanism remain unclear. In this proposal, we propose to investigate the important role of exosomes in mediating inflammatory effect of CNMs in macrophages and further study the molecular mechanism based on the exosomes-mediated signaling pathways. This project will help to reveal the novel molecular mechanism of CNMs-induced inflammatory effect on the immune cells. Firstly, we will study the intracellular accumulation and distribution of CNMs in macrophages by gel electrophoresis and TEM, respectively. Moreover, we will detect the ability of exosomes secretion stimulated by three types of CNMs using nanoparticle tracking analysis (NTA). Secondly, by using the P2X7 receptor knockout macrophages, we can clarify the role of P2X7R signaling pathway in the exosomes secretion. In addition, the internalization pathways of exosomes can be determined by using endocytosis inhibitors and flow cytometry analysis. Thirdly, proteomics will be used to identity the key inflammatory molecules in exosomes of macrophages exposed to CNMs. At last, cell co-culture model and animals were utilized to confirm the inflammatory effect of exosomes released by CNMs-stimulated macrophages. A better understanding of multiple inflammatory mechanisms is important for hazard assessment and could allow their safety by design and production of new medical and environmental applications.

纳米材料安全性研究远远滞后于纳米技术的快速发展，这极大地限制了纳米材料的应用。因此有必要深入探索纳米材料的安全性。目前，碳纳米材料（CNMs）的广泛应用使得人们极为关注其潜在的健康危害，尤其是炎性反应。近年来，外泌体作为新型的细胞通讯介质，已被证实能够通过装载的炎性分子介导靶体的炎症反应，但其作用机制尚不清楚。本课题拟研究外泌体介导的碳纳米材料炎性反应及分子机制。通过聚丙烯酰胺凝胶电泳及透射电镜考察三种CNMs在细胞内的累积及分布；通过纳米示踪分析（NTA）检测CNMs刺激细胞释放外泌体的能力；通过应用P2X7受体基因敲除细胞，研究P2X7受体对外泌体分泌的调控作用；通过蛋白组学方法鉴定外泌体中关键炎性分子；应用细胞共培养及动物体系，进一步研究CNMs刺激巨噬细胞释放的外泌体介导的炎症反应。在此基础上，阐释CNMs导致炎性反应的新型机制，揭示外泌体对介导CNMs炎性反应的重要作用。

纳米材料安全性研究远远滞后于纳米技术的快速发展，这极大地限制了纳米材料的应用。因此有必要深入探索纳米材料的安全性。目前，碳基纳米材料（CNMs）的广泛应用使得人们极为关注其潜在的健康危害，尤其是炎症反应。近年来，外泌体作为新型的细胞通讯介质，已被证实能够通过装载的炎症分子介导靶体的炎症反应，但其作用机制尚不清楚。本课题研究外泌体介导的碳基纳米材料炎症反应及分子机制。通过聚丙烯酰胺凝胶电泳及透射电镜发现巨噬细胞能够摄入大量碳基纳米材料并分布于囊泡中；通过纳米示踪分析（NTA）检测不同理化性质碳基纳米材料刺激细胞释放外泌体的能力，发现小尺寸碳基纳米材料能够刺激巨噬细胞释放更多外泌体；通过应用P2X7受体抑制剂，首次揭示P2X7受体及下游信号通路对外泌体分泌的调控作用（伴随微管重排）；通过细胞共培养和蛋白组学方法，阐明了外泌体激活受体巨噬细胞TLR-4—NF-kB炎症信号通路，进而刺激巨噬细胞释放大量炎症因子（TNF-a、IL-1β和Muc5b）；应用回输技术及动物体系，进一步揭示碳基纳米材料刺激巨噬细胞释放的外泌体能够介导体内炎症反应。基于以上事实，我们首次阐释了碳基纳米材料导致炎症反应的新型机制，这对于纳米材料的风险评价至关重要。此外，该项目的实施为设计、生产和使用更安全的的纳米材料提供了重要理论依据。