单壁碳纳米管的免疫毒理和毒理基因组学分子机制研究

项目名称： 单壁碳纳米管的免疫毒理和毒理基因组学分子机制研究

项目编号： No.20807054

项目类型： 青年科学基金项目

立项/批准年度： 2009

项目学科： 无线电电子学、电信技术

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： 19万元

中文摘要： 碳纳米管在电子技术、生物医学以及环境治理等领域存在着广泛的应用前景，同时需要关注其对人类和环境的不良健康效应。本课题系统研究了单壁碳纳米管（SWCNTs）对于巨噬细胞的毒性效应、致毒分子机理、及其对细胞器和细胞生理功能的影响。分别以小鼠巨噬细胞RAW264.7和原代腹腔巨噬细胞为模型，研究了SWCNTs的免疫细胞毒性及分子机制。首先用混酸处理得到具有良好水分散性的CNTs（AF-SWCNTs）。利用荧光示踪以及TEM发现其在细胞内大多分布于细胞质，在溶酶体中大量存在。荧光探针和基因表达分析发现AF-SWCNTs对蛋白酶体和线粒体也有损伤作用，并抑制巨噬细胞的吞噬和辅助细胞功能，同时诱导IFN-r和TNF的分泌促使T细胞向Th1细胞分化。用基因芯片技术揭示了受AF-SWCNTs影响的全基因组表达谱的变化，发现10-50 ug/mL AF-SWCNTs 影响了细胞的线粒体、蛋白酶体等细胞器以及细胞周期/凋亡等多个信号通路的基因表达。本课题首次系统研究了SWCNT本身对免疫系统的损伤作用及其分子机制，排除了杂质（如金属杂质等）的干扰，为SWCNT的生物应用安全性评价提供了客观的科学依据。

中文关键词： 单壁碳纳米管;巨噬细胞;毒性机制;细胞功能分析;基因芯片

英文摘要： Carbon nanotubes have attracted great interests in various applications, such as electronics, biomedical and environmental protection in recent decades. The potential adverse effects on human and environment health are a critical factor before such nanomaterials can be used for biomedical purposes, especially in vivo applications. In this project, mouse cell line RAW264.7 and primary peritoneal macrophages were used as models for studying the toxic effects and toxic mechanisms of SWCNTs. Firstly, SWCNTs were treated with mixed acids resulting in stable aqueous CNTs solution. After characterizations by TEM and Zeta potential, SWCNTs were used to expose to cells and the distribution of CNTs within cells were analyzed by using fluorescent probes and ultra-thin cell section. Results found that most CNTs were located within cell cytosol, including lysosomes. Exposure of SWCNTs can cause serious damages to organelles including proteasome and mitochondrial, as evidenced by Q-PCR and JC-1 assay. The consequences of such effects have been determined as the inhibition of phagocytic function and accessory cell function of macrophages. In addition, SWCNTs induced the expression of cytokines (including IFN-r and TNF) that preferentially promote the differentiation of naive T cells into Th1 type, suggesting the risk of Th1 type diseases (e.g. autoimmunity and inflammation) upon SWCNT exposure. Further study using microarray on the gene expression profiles revealed that multiple pathways were modulated by SWCNTs including cell apoptosis/cell cycle, ribosome, proteasome, mitochondrial and inflammation.

英文关键词： SWCNT; Macrophages; Toxic mechanism; Functional analysis; Microarray