纳米氧化锌对小鼠腹腔巨噬细胞Ana-1的毒性作用及机制

摘要

目的 研究纳米氧化锌(ZnO-NP)对小鼠腹腔源巨噬细胞(Ana-1)的毒性作用及机制.方法 ZnO-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ZnO-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 ZnO-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);ZnO-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;ZnO-NP 40,80和160 mg·L-1组细胞摄取的ZnO-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ZnO-NP 10 mg·L-1引起的细胞存活率下降(P＜0.05).螯合锌离子后,ZnO-NP对Ana-1细胞的毒性明显降低(P＜0.05).结论 ZnO-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 (ZnO-NPs) on murine macrophage Ana-1 cells and the mechanism.METHODS Ana-1 cells were incubated with ZnO-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 ZnO-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 ZnO-NP 2.5,5,10 and 20 mg· L-1 decreased 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 ZnO-NP 20 mg· L-1.Intracellular uptake of ZnO-NP was increased after Ana-1 cell incubated with ZnO-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-1 significantly increased the cell viability decreased by ZnO-NP 20 mg· L-1 (P＜0.05).Chelating free zinc ions significantly mitigated ZnO-NP induced cell toxicity (P＜0.05).CONCLUSION Cytotoxicity and apoptosis of Ana-1 cells induced by ZnO-NP might be related to intracellular uptake of ZnO-NP and release of zinc ions.