纳米银对中国仓鼠肺成纤维细胞的细胞毒性和遗传毒性

Cytotoxicity and genotoxicity of silver nanoparticles on the Chinese hamster lung fibroblast cell line

目的:研究纳米银对中国仓鼠肺成纤维细胞(CHL)的细胞毒性和遗传毒性并探讨其作用机制。方法:以梯度浓度(2.5、5.0、10.0、20.0、40.0和80.0μg/m L)纳米银染毒CHL细胞,MTT比色法计算IC5 0以评估纳米银的细胞毒性水平。利用PI染色分析细胞周期变化并计算细胞增殖指数;Annexin V-FITC和PI双染检测细胞凋亡和坏死发生率,同时观察细胞染毒后的形态变化,记录前向角散射(FSC)和侧向角散射(SSC)信号;Giemsa染色中期分裂相涂片分析染色体畸变发生率。结果:纳米银毒性水平与纳米银浓度呈正相关(r=0.804,P<0.05),IC 50为21.68μg/m L。22.0μg/m L纳米银与CHL细胞接触24 h后,细胞形态发生明显改变;细胞群SSC信号显著增强,为细胞摄入纳米银提供间接证据;细胞周期在G2/M期被捕获,对G0/G1期、S期以及细胞增殖指数变化情况影响显著(P<0.01);22.0μg/m L纳米银染毒CHL 24 h后凋亡发生率与对照组差异明显(P<0.01);各浓度纳米银诱导染色体结构畸变率均大于5%(P<0.01),中高浓度四倍体发生率达到溶剂对照组的10倍以上(P<0.01)。结论:纳米银对CHL细胞有显著的细胞毒性和遗传毒性,线粒体功能受损、细胞凋亡在纳米银的细胞毒性和遗传毒性中起到重要作用。

OBJECTIVE： To study the cyto-and genotoxicities of silver nanoparticles （silver-NPs） on Chinese hamster lung fibroblast cell line （CHL） and explore their possible mechanisms. METHODS：CHLs were exposed to gradient concentrations of silver-NPs 2.5,5.0,10.0,20.0,40.0 and 80.0μg/mL,then MTT assay was employed to reveal the IC50 which was used to evaluate the cytotoxicity potential of silver-NPs. Cell cycle analysis was performed with PI dye,and proliferation indexes were calculated as well. Apoptosis was detected with Annexin V FITC-PI dye,morphology changes after exposure and forward scatter channel （FSC） and side scatter channel（SSC） were recorded simultaneously. Chromosomal aberrations were analyzed by reading slides with metaphases stained by Giemsa. RESULTS：Cytotoxicity on CHL was positively associated with the concentration of silver-NPs （r=0.804,P〈0.05）, IC50 was 21.68 μg/mL. Morphology changes were clearly visible after CHLs were treated with 22.0 μg/mL silver-NPs for 24 h. Significantly enhanced SSC signal existed in CHL populations after periods of exposure to silver-NPs,which implied uptake of silver-NPs. CHL cycle was found to be arrested in G2/M phase,and cell proliferation index as well as G0/G1 phase,S phase were significantly changed compared with vehicle control （P〈0.01）. Furthermore,apoptosis was found to be significantly increased after 22.0 μg/mL silver-NPs exposure for 24 h （P〈0.01）. Silver-NPs exposure also induced CHL chromosome structure aberration with more than 5% incidence at all tested concentrations （P〈0.01）. Tetraploid in high and middle concentrations-treated groups were 10 times higher than the vehicle control（P〈0.01）. CONCLUSION：Silver-NPs showed significant cytotoxicity and genotoxicity in CHL,where mitochondrial dysfunction and apoptosis might play important roles.