几种碳纳米材料诱导的细胞自噬效应及其毒性作用机制研究

项目名称： 几种碳纳米材料诱导的细胞自噬效应及其毒性作用机制研究

项目编号： No.21477146

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 化学工程及工业化学

项目作者： 万斌

作者单位： 中国科学院生态环境研究中心

项目金额： 90万元

中文摘要： 碳纳米材料的广泛应用使得人们极为关注其环境和健康危害。近年来，细胞自噬性死亡成为生命科学的一个研究热点，可能是碳纳米材料的一种潜在致毒机制。本课题拟研究三种典型碳纳米材料的细胞毒性、自噬诱导及其在细胞毒性机制中的作用。通过一系列细胞活性检测方法考察不同纳米材料的细胞毒性，同时采用电镜观察、自噬检测细胞模型和生物化学方法研究细胞内自噬体的累积；进一步通过考察p62蛋白丰度和对溶酶体的影响来研究碳纳米材料对自噬流的干扰作用，以阐明自噬发生的作用机制。在此基础上，采用抑制剂和基因沉默方法对自噬进行阻断，考察由此引起的细胞毒性变化，以研究自噬的发生在细胞毒性机制中的作用；通过对比不同形态和表面基团的碳纳米材料，研究影响纳米材料自噬诱导能力的因素。阐明碳纳米材料诱导的细胞自噬与细胞毒性的关系将极大地促进对碳纳米材料致毒机制的理解，为碳纳米材料的应用提供新的视野，并对其安全性做出全面客观的科学评价。

中文关键词： 碳纳米材料；细胞自噬；自噬流；毒性作用机制；细胞毒性

英文摘要： The wide use of carbon nanomaterials (CNMs) in environmental and medical fields has attracted a great deal of concerns on their potential adverse effects on environment and human health. Autophagic cell death has recently become a hot topic in life science, and might be a potential toxic mechanism of CNMs besides apoptosis. In this proposal, we proposed to study the cytotoxicity, autophagy induction and its roles in the toxic mechanisms of three typical CNMs including C60, SWCNTs, and GOs. First, the cytotoxicity will be examined by using a variety of cell viability assays based on different principles and detection procedures including WST-1, flow cytometry analysis and Live/Dead fluorescent staining, to avoid potential interference of CNMs. Simultaneously, the autophagosome accumulation can be detected through TEM, transfection cell model and biochemistry methods. Furthermore, the influence of CNMs on autoflux can be studied by monitoring the abundance of p62 protein and lysosomes, to illustrate the mode of autophagy induction. After that, specific inhibitors and RNAi will be adopted to inhibit CNM-induced autophagy and the subsequent influence on cell viability will be studied to reveal the roles of autophagy in toxic mechanisms of CNMs. At last, the autophagy induction capabilities of CNMs with various shapes and surface functional groups will be compared to shed light on the influential factors of autophagy induction. Study of the relationship between autophagy induction and cytotoxicity will greatly facilitate a better understanding of the toxic mechanisms of CNMs, bring new insights on potential CNMs applications, and provide data for comprehensive and scientific hazard assessment on CNMs.

英文关键词： Carbon nanoparticles;Cell autophagy;Autoflux;Toxic mechanisms;Cytotoxicity