陆杰， 应佳丽， 张婷^*

（  东南大学公共卫生学院，环境医学工程教育部重点实验室，江苏省生物材料与器件重点实验室，南京 210009；  ）

摘要： 目的：探讨γ-Fe2O3和纳米氧化锌颗粒（ZnO NPs）对人肝癌细胞（HepG2）的毒性作用及其影响因素，为评价纳米金属氧化物对生物体的肝毒性提供基本的科学依据。方法：利用透射电镜和马尔文激光粒度仪表征两种纳米材料的尺寸，水合粒径和Zeta电位；利用原子吸收光谱法和火焰原子吸收法分别测定培养基中金属氧化物的含量和细胞内铁元素和锌元素的含量；采用针对铁离子和锌离子的荧光探针方法检测细胞内铁离子和锌离子的含量；MTT法检测两种纳米材料对HepG2细胞活性的影响；Annexin-V/PI双染法检测细胞凋亡的情况；利用荧光探针DCFH-DA检测细胞活性氧（ROS）的含量。结果：电镜表征结果显示Fe2O3和ZnO的粒径分别为8.70±2.6nm和22.71±4.70nm；培养基中金属离子随着染毒浓度增高而升高，但随时间延长而减少；细胞内两种元素含量则具有正向的时间和剂量依赖性关系。MTT结果显示两种金属氧化物均具有剂量依赖性关系，并且同等浓度下ZnO NPs抑制率比Fe2O3高，这与细胞凋亡的结果相一致。ROS检测结果显示ZnO组细胞ROS的含量具有明显剂量依赖性关系，而Fe2O3组ROS含量随剂量增大而增大，但趋势不如ZnO组明显。结论：两种金属纳米材料对HepG2细胞的毒性的可能原因是进入细胞后释放金属离子导致ROS水平升高所致，造成两种金属纳米材料毒性差异的原因是组成化学物的化学性质不同，化学性质活泼者毒性越大，如更容易释放金属离子，而粒径等因素并不是主要因素。
关键词： 纳米毒理学；金属氧化物；毒性作用；影响因素

Lu Jie， Ying JiaLi， Zhang Ting^*

（  Key Laboratory of Environmental Medicine Engineering, Jiangsu Key Laboratory for Biomaterials and Devices, School of Public Health, Southeast University, Nanjing 210009；  ）

Abstract： Objective: To investigate the toxic effects of γ-Fe2O3 and ZnO nanoparticles (ZnO NPs) on human hepatocellular carcinoma cells (HepG2), and to provide basic scientific evidence for evaluating hepatotoxicity of nano-metal oxides on organism. Method: The size, hydration size and zeta potential of the two nanomaterials were characterized by transmission electron microscopy and Malvern laser particle size analyzer. The contents of metal oxides and intracellular iron contents were determined by atomic absorption spectrometry and flame atomic absorption spectrometry, respectively. The contents of iron and zinc in the cells were measured by the fluorescent probe method. The MTT assay was used to detect the effect of the two kinds of nano-materials on the activity of HepG2 cells. Annexin-V / PI double staining (ROS) was detected by fluorescent probe DCFH-DA. Results: The results of electron microscopy showed that the particle size of Fe2O3 and ZnO was 8.70 2.6nm and 22.71±4.70nm, respectively. The metal ions in the medium increased with the increase of the concentration of Fe2O3 and ZnO, but decreased with the prolongation of time. And the content was in a time-dependent and dose-dependent manner. MTT results showed that both metal oxides had a dose-dependent relationship, and the inhibitory rate of ZnO NPs was higher than that of Fe2O3 at the same concentration, which was consistent with the results of apoptosis. The results of ROS showed that the content of ROS in the cells of ZnO group was dose-dependent, while the content of ROS in Fe2O3 group increased with the dose increasing, but the trend was not as obvious as in ZnO group. Conclusion: The toxicity of the two metal nanomaterials to HepG2 cells was due to the release of metal ions into the cells and the increase of ROS level. The difference of toxicity between the two metal nanomaterials were attributed to the different chemical properties of the chemical constituents, The greater the toxicity of lively, such as easier to release metal ions, and particle size and other factors are not the main factors.
Keywords： Nano Toxicology; metal oxides; toxicity; Influencing factors

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