表面不同修饰的两种多壁碳纳米管引起RAW264.7细胞蛋白质差异表达

Two types of MWNTs with different surface modifications induce differential expression of proteins in RAW264.7 cells

目的:比较表面不同修饰的两种多壁碳纳米管[经酸化处理的多壁碳纳米管(acid-treated multi-walled carbon nanotubes,aci-MWNTs)和牛磺酸表面修饰的多壁碳纳米管(taurine-modified multi-walled carbon nanotubes,tau-MWNTs)]引起小鼠巨噬细胞RAW264.7细胞蛋白质表达的差异。方法:两种碳纳米管均以20mg/L的剂量,24h的时间染毒细胞,并设置空白对照组;运用尿素裂解、冰浴超声的方法破碎细胞,提取细胞全蛋白,运用二向凝胶电泳分离细胞全蛋白,寻找银染凝胶图像上表达差异蛋白点,对差异蛋白行质谱分析鉴定,在蛋白水平上探究两种MWNTs对细胞作用的机制。结果:两种碳纳米管处理的细胞,表达有显著差异的蛋白点共13个,功能包括凋亡相关、钙结合、细胞周期相关、DNA合成、蛋白质折叠和能量代谢等方面,并与本课题组前期研究观察到的MWNTs对细胞凋亡诱导及线粒体损伤等细胞毒性结果相吻合。结论:两种MWNTs均能引起RAW264.7细胞蛋白表达的变化,表面不同修饰的MWNTs作用有差异,高通量蛋白质组学方法有望用于碳纳米管生物学效应及毒性机制研究。

Objective：To compare the different expression of protein in RAW264.7 macrophage cells induced by two types of MWNTs（multi-walled carbon nanotubes）with different surface modifications（acid-treated MWNTs and tau-MWNTs modified by taurine）.Methods：Treating cells with both types of MWNTs in 20 mg/L and 24 h,with a blank-control group set.Cells are lysed by using urea and by ultrasonicating in ice bath,then total proteins of cells are extracted.Using two-dimensional gel electrophoresis to separate total proteins of cells,searching for the differential expressed protein spots on the images of the gels with silver staining.Identifying the differentially expressed proteins via mass spectrometry,and studying the mechanism of effects on cells imposed by two types of MWNTs at protein level.Results：There are 13 spots of protein with notably differential expression among three treated groups（including blank controls）.Their functions involve apoptosis-related,calcium-binding,cell-cycle related,DNA synthesis,folding of proteins,and energy metabolism,etc.The results are consistent with our previous studies about the cytotoxicity of Both types of MWNTs including induction of apoptosis and mitochodira damage.Conclusion：Both two types of MWNTs could induce alteration of protein expression in RAW264.7 cells.With different surface modifications,they imposed different effects.High throughout proteomics could be applied in toxicity assessment and mechanism investigation about carbon nanotubes.