纳米银的线粒体毒性效应与作用机制研究

Silver nanoparticles (Ag NPs) are one of the most widely used nanomaterials in the medical, health and personal products. The environmental and health exposure risks from Ag NPs are increasing with the explosive Ag NPs-based goods in the market. Though the toxicity of silver nanoparticles has been widely studied, the knowledge of toxicity and health risk of Ag NPs in environment or life is still largely unknown. Our previous results showed that Ag NPs could obviously induce the release of cytochrome c from mitochondria. Therefore, on this basis, this project will focus on the toxicity of low dose of silver nanoparticle on mitochondria function and reveal related mechanism. First, utilizing the several cell lines, the toxicity, location and distribution of Ag NPs with various physicochemical properties in the mitochondria will be studied, especially the intracellular species of Ag NPs (i.e. Ag NPs and Ag+) and their proportion will be analyzed. Then the effects of Ag NPs on the structure and function of mitochondria will be evaluated to reveal the molecular mechanism involved in the toxicity of Ag NPs. Finally, the toxic effects of Ag NPs due to mitochondria lesion in cells and animals will be studied. We believe this project will provide the important scientific bases and valuable information to understand the toxicity mechanism of Ag NPs.

纳米银是医疗卫生和日常生活等领域应用最广泛的纳米材料，其环境健康风险也备受关注。尽管针对纳米银的毒性已开展了大量研究，然而，目前对于纳米银暴露的毒性与健康危害认识较少。申请人在前期研究中发现纳米银能够与线粒体相互作用并影响其生理功能。基于已有的研究基础，申请人拟在以下方面开展纳米银的线粒体损伤与毒性效应研究，包括：研究纳米银在线粒体上的分布特征和赋存状态信息，特别是纳米银暴露的“颗粒效应”与“离子效应”对毒性效应的贡献；研究纳米银诱导线粒体损伤及分子机制；研究米银通过线粒体损伤诱导的生物毒性效应。通过以上系统研究，深入了解和探明纳米银的毒性效应机制。

（1）开发了一种基于共聚焦显微镜的散射光成像新技术，实现了对活细胞内非标记纳米颗粒和荧光标记生物分子的同时成像。在共聚焦显微镜荧光通路模式下对胞内纳米颗粒的散射光进行成像分析（散射光成像，SLi）。研究表明，借助于光栅分离波长，可以用不同通道同时收集纳米颗粒的散射光和荧光标记生物分子的荧光信号，从而实现两者的同步观测。SLi成像模式可以分辨胞内20-100纳米不同尺寸的纳米银颗粒（空间分辨率10纳米），并实现胞内纳米颗粒24小时的超长连续观察。该方法可在单颗粒水平研究纳米颗粒的跨膜运输、胞内分布、转化过程以及和生物大分子的相互作用。. （2）以线粒体为研究重点，考察了nAg对细胞线粒体超氧化物水平、线粒体膜电势、线粒体膜孔通透性、细胞色素C释放量及促凋亡酶caspase 3/7活性的影响。证实了细胞摄入的nAg以粒子的形式存在；对线粒体状态研究表明，nAg和Ag+引起线粒体超氧化物上升并降低线粒体膜电势，但只有nAg颗粒可以引起细胞线粒体膜孔打开，释放出细胞色素C，激活下游凋亡酶caspase 3/7活性。. （3）研究发现超长AgNWs可以干扰细胞正常分裂过程，导致细胞周期停滞并生成大量的巨型多核细胞。这种干扰效应与超长AgNWs的暴露剂量和直径紧密相关。进一步研究发现超长AgNWs可以显著地促进有丝分裂过程中关键调控蛋白Aurora A、p-Histone 3 (ser10)、p-MLC和Myosin IIb表达量升高。同时，实时成像观察结果表明超长AgNWs能够在分裂末期的胞质分裂区聚集，通过直接阻碍收缩环的内缩而导致两个子细胞胞质分离失败，重新融合后形成多核细胞。