纳米氧化锌对小鼠腹腔巨噬细胞Ana-1的毒性作用及机制

Cytotoxicity and mechanism of zinc oxide nanoparticles on murine macrophage Ana-1 cells

目的研究纳米氧化锌(Zn O-NP)对小鼠腹腔源巨噬细胞(Ana-1)的毒性作用及机制。方法Zn O-NP 2.5~160 mg·L^(-1)与Ana-1细胞作用24 h后,MTT法检测Ana-1细胞的存活率;吖啶橙-溴化乙锭(AO-EB)染色及流式细胞术观察Ana-1细胞凋亡;流式细胞术检测细胞对Zn O-NP的摄取;锌离子荧光探针测定细胞内的锌离子;乙二胺四乙酸(EDTA)螯合锌离子,检测细胞存活率。结果 Zn O-NP在2.5,5,10和20 mg·L^(-1)浓度范围内能够浓度依赖性地抑制Ana-1细胞存活(r=0.905,P<0.05);Zn O-NP20 mg·L^(-1)组细胞存活率为细胞对照组的27.9%,细胞出现明显的凋亡样改变;Zn O-NP 40,80和160 mg·L^(-1)组细胞摄取的Zn O-NP比细胞对照组显著增加(P<0.05);EDTA 2.5 mmol·L^(-1)能够明显降低细胞内的自由锌离子,改善Zn O-NP 10 mg·L^(-1)引起的细胞存活率下降(P<0.05)。螯合锌离子后,Zn O-NP对Ana-1细胞的毒性明显降低(P<0.05)。结论 Zn O-NP可浓度依赖性增加Ana-1细胞的毒性,引起凋亡样改变,其毒性可能与其进入细胞并释放锌离子有关。

OBJECTIVE To study the toxicity of zinc oxide nanoparticles（Zn O-NPs） on murine macrophage Ana-1 cells and the mechanism. METHODS Ana-1 cells were incubated with Zn O-NP（2.5-160 mg·L-1）. Cell viability was investigated by MTT assay. The integrity of cell membrane was investigated by acridine orange-ethidium bromide（AO-EB） staining. The intracellular uptake of Zn O-NP and the percentage of sub-G1 of Ana-1 cells were detected by flow cytometry. Zinc ions were determined by fluorescent probe. The change of cell viability was studied after chelating zinc ions with ethylene diamine tetraacetic acid（EDTA）. RESULTS Zn O-NP 2.5, 5, 10 and 20 mg·L-1decreased cell viability of Ana-1 cells（r=0.905, P〈0.05） in a concentration-dependent manner. The cell viability was decreased to 27.9% after exposure to Zn O-NP 20 mg·L-1. Intracellular uptake of Zn O-NP was increased after Ana-1cell incubated with Zn O-NP at concentrations ranging from 40 to 160 mg·L-1（P〈0.05）. There were obvious free zinc ions in the cells. EDTA 2.5 mmol·L-1significantly increased the cell viability decreased by Zn O-NP 20 mg·L-1（P〈0.05）. Chelating free zinc ions significantly mitigated Zn O-NP induced cell toxicity（P〈0.05）. CONCLUSION Cytotoxicity and apoptosis of Ana-1 cells induced by Zn O-NP might be related to intracellular uptake of Zn O-NP and release of zinc ions.