摘要目的 探讨纳米氧化锌(ZnO)对U937细胞的毒性及机制.方法 对4种不同粒径的ZnO(直径分别为10、30、60、500nm)进行详细表征.通过台盼蓝拒染实验、噻唑蓝(MTT)实验检测各种粒径ZnO在不同浓度时(浓度分别为12、120、240、600、1200 μmol/L)对U937细胞存活率和细胞活力的影响;利用锌离子探针监测细胞内锌离子浓度变化;利用透射电子显微镜观察细胞超微结构及其对ZnO的吞噬情况.结果 4种粒径ZnO均呈棒状,属于闪锌矿,六方晶系,样品纯度>99%,比表面积变化规律与粒径相吻合.浓度12～600 μmol/L,4种ZnO染毒组细胞相对活力[ZnO-n10为:(97±19)%、(91 ±4)%、(24 ±4)%、(15±2)%;ZnO-n30为:(111±24)%、(81±3)%、(24 ±2)%、(27±8)%;ZnO-n60为:(105±11)%、(73±20)%、(43±11)%、(28±14)%;ZnO-μm为:(88±16)%、(62±7)%、(22±4)%、(13±5)%]均随着染毒浓度的增加而降低(r值分别为:0.965、0.979、0.998、0.992;对应的相关系数统计检验值t值分别为:19.8、25.3、76.3、40.9,P值均<0.05).浓度12～1200 μmol/L,4种ZnO染毒组细胞存活率[ZnO-n10为:(98±1)%、(67±2)%、(59±7)%、(13±13)%、(5±4)%;ZnO-n30为:(98±1)%、(97±2)%、(50±3)%、(20±14)%、(7±2)%;ZnO-n60为:(97±2)%、(88±5)%、(48±10)%、(12±5)%、(4±1)%;ZnO-μm为:(96±1)%、(76 ±3)%、(58±3)%、(19 ±5)%、(20±10)%]随着浓度的增加而降低(r值分别为:0.982、0.956、0.972、0.980;对应的相关系数统计检验值f值分别为:19.3、12.1、15.6、18.5,P值均<0.05).ZnO-n30引起的细胞内锌离子荧光探针的荧光强度的增加值(121±11)与等锌量ZnAc2引起的升高值(平均强度132±14)一致(F=1.6,P>0.05).200 μmol/L ZnO-n30或ZnAc2孵育3 h后,ZnO-n30使高锌含量细胞占总细胞数的(87.6±2.6)%,ZnAc2组为(86.9±3.2)%,二者差异无统计学意义(F=1.5,P>0.05).结论 纳米ZnO对U937细胞产生严重的细胞毒性,溶解产生锌离子是主要机制.

abstractsObjective To investigate the cytotoxicity and its mechanism of ZnO nanoparticles on human leukemic monocyte lymphoma cell line U937. Methods Four different size ZnO (10, 30, 60,500 nm) were carefully characterized. The survival rate and viability were measured by trypan blue assay and MTT assay for each size ZnO particles at different concentrations (12,120,240,600,1200 μmol/L). The zinc probe, Fluozin-3, was used to detect the intracellular free zinc. Transmission electron microscopy was adopted to observe the cellular ultrastructure and the uptake of ZnO. Results All four kinds of ZnO were rod shape,with a purity of >99.9 wt% ,and they were classified as zincite phase crystal and their surface areas were in accordance with the sizes. The viability (ZnO-n10:(97 ± 19)%, (91 ±4)%, (24±4)%,(15±2)%; ZnO-n30:(111 ±4)%,(81 ±3)%,(24 ±2)%,(27 ±8)%; ZnO-n60:(105 ±11)%,(73 ±20)%,(43 ±11)%,(28±14)%; ZnO-μm: (88 ±16)%,(62 ±7)%,(22 ±4)%,(13 ±5)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 μmol/L (r values were 0. 965,0. 979,0. 998,0. 992, and the t values were 19. 8,25. 3,76. 3,40.9, respectively, P <0.05). The liability (ZnO-n10:(98 ±1)% ,(67 ±2)% ,(59 ±7)% ,(13 ±13)% ,(5 ±4)%; ZnO-n30:(98±1)%,(97 ±2)%,(50±3)%,(20±14)%,(7 ±2)%; ZnO-n60:(97 ±2)%,(88 ±5)%,(48 ±10)%,(12 ±5)%,(4 ±1)%; ZnO-μm: (96 ±1)% ,(76 ±3)%,(58 ±3)%,(19 ±5)%,(20 ±10)%) of cells exposed to ZnO decreased with the increasing of the concentration of ZnO from 12 to 600 μnol/L (r valued at 0.982,0.956,0.972,0.980, and the t valued at 19.3, 12. 1, 15.6, 18.5,respectively,P < 0. 05). The increase of the zinc concentration showed by the zinc fluorescence probe was 121 ± 11 ,which was similar to the fluorescence of cells treated with ZnAc2 (132±14, F = 0. 6, P > 0. 05) at the Zn-equivalent concentration. There was no statistic difference for the percents of high zinc content cells in total cells exposed to ZnO-n30 (87. 6 ± 2. 6) % and these exposed to ZnAc2 (86. 9 ± 3. 2) % (F = 1.5,P >0. 05). Conclusion ZnO nanoparticles are highly cytotoxic to U937 cells and the solubilization of ZnO is the main toxicological mechanism.