纳米二氧化硅颗粒对血管内皮细胞的毒性及其凋亡诱导作用

doi:10.13481/j.1671-587x.20150304

吉林大学学报(医学版) ›› 2015, Vol. 41 ›› Issue (03): 454-459.doi: 10.13481/j.1671-587x.20150304

纳米二氧化硅颗粒对血管内皮细胞的毒性及其凋亡诱导作用

夏银叶^1,2, 李艳博^2,3, 牛丕业^1,2, 王晖⁴, 王翼飞⁴, 梁宝璐⁴, 赵峰^1,2, 郭彩霞^1,2, 孙志伟^2,3

1. 首都医科大学公共卫生学院劳动卫生与环境卫生学系, 北京 100069;
2. 首都医科大学环境毒理学北京市重点实验室, 北京 100069;
3. 首都医科大学公共卫生学院卫生毒理与卫生化学学系, 北京 100069;
4. 首都医科大学公共卫生学院公共卫生与预防医学教学实验中心, 北京 100069

收稿日期:2013-09-25 发布日期:2015-08-01
通讯作者: 郭彩霞,讲师(Tel:010-83911774,E-mail:guocx@ccmu.edu.cn);赵峰,副教授,硕士研究生导师(Tel:010-83911505,E-mail:feng_zhao66@sina.com) E-mail:guocx@ccmu.edu.cn;feng_zhao66@sina.com
作者简介:夏银叶(1984-),女,内蒙古自治区乌兰浩特市人,在读医学硕士,主要从事环境有害因素对健康危害方面的研究。
基金资助:
国家自然科学基金资助课题(81102095,81202242,81172704);北京高等学校青年英才计划项目资助课题(YETP1670);首都医科大学科技处临床流行病学北京市重点实验室开放课题(2013LCLB04)

Toxicity of silica nanoparticles on vascular endothelial cells and its induction effect on apoptosis

XIA Yinye^1,2, LI Yanbo^2,3, NIU Piye^1,2, WANG Hui⁴, WANG Yifei⁴, LIANG Baolu⁴, ZHAO Feng^1,2, GUO Caixia^1,2, SUN Zhiwei^2,3

1. Department of Occupational and Environmental Health, School of Public Health, Capital Medical University, Beijing 100069, China;
2. Beijing Key Laboratory of Environmental Toxicology, Capital Medical University, Beijing 100069, China;
3. Department of Toxicology and Sanitary Chemistry, School of Public Health, Capital Medical University, Beijing 100069, China;
4. Experimental Teaching Center for Public Health and Preventive Medicine, School of Public Health, Captial Medical University, Beijing 100069, China

Received:2013-09-25 Published:2015-08-01

摘要/Abstract

摘要：

目的:研究纳米二氧化硅(SiO₂)颗粒的血管内皮细胞毒性和可能的作用机制,为探讨纳米SiO₂颗粒毒性效应及安全性评价提供参考依据。方法:将体外培养的人脐静脉内皮细胞(HUVECs)随机分为对照组和不同浓度纳米SiO₂颗粒暴露组,暴露浓度分别为12.5、25.0、50.0及100.0 mg·L^-1。采用透射电镜(TEM)观察纳米SiO₂颗粒粒径、形貌及分散性。细胞处理24 h后,电感耦合等离子体原子发射光谱(ICP-AES)法测定细胞中硅水平;MTT法测定细胞活力;乳酸脱氢酶(LDH)释放法检测细胞膜的完整性;DCFH-DA荧光探针标记激光共聚焦显微镜观察细胞中活性氧(ROS)水平;DAPI染色荧光显微镜下观察细胞核形态;Annexin Ⅴ/PI双染标记流式细胞术(FCM)检测细胞凋亡率;罗丹明123线粒体试剂盒检测细胞线粒体膜电位。结果:TEM观察,纳米SiO₂颗粒分布均匀,大小一致,颗粒呈球形,分散性好,未发生聚集。用Image J 软件计算得到颗粒平均粒径为(57.66 ± 7.30)nm。与对照组比较,纳米SiO₂颗粒作用于HUVECs后,细胞活力下降(P<0.05),细胞中硅水平和培养液中LDH活性增加(P<0.05),细胞中ROS水平升高,线粒体膜电位下降,甚至发生细胞凋亡。上述细胞效应均表现为随颗粒作用浓度的增加而逐渐明显,呈现一定的剂量依赖关系。结论:纳米SiO₂颗粒具有血管内皮细胞毒性,可诱导ROS生成和氧化应激,从而破坏细胞膜、损伤线粒体,最终引发细胞凋亡。

关键词: 纳米二氧化硅, 细胞毒性, 细胞凋亡, 线粒体

Abstract:

Objective To explore the cytotoxicity of silica nanoparticles(SiNPs) on the vascular endothelial cells and possible mechanism, and to provide reference for the study on the toxic effects and safety evaluation of SiNPs. Methods The in vitro cultured human umbilical vein endothelial cells (HUVECs) were randomly divided into control and SiNPs exposure groups at the concentrations of 12.5, 25.0, 50.0, and 100.0 mg·L^-1, respectively.Transmission electron microscope (TEM) was used to determine the particle size, morphology and dispersion of SiNPs.After 24 h exposure, inductively coupled plasma atomic emission spectroscopy (ICP-AES) was used for the measurement of intracellular silicon level, MTT assay for the determination of cell viability, lactate dehydrogenase (LDH) release assay for membrane integrity, laser confocal microscope with DCFH-DA fluorescence probe for intracellular reactive oxygens pecies (ROS) level, DAPI staining under fluorescence microscope for the observation of nuclear morphology, flow cytometry (FCM) with Annexin Ⅴ and PI double staining for the measurement of apoptosis, and Rhodamine 123 kits for mitochondrial membrane potential (MMP). Results The TEM Results showed that SiNPs were mostly spherical with uniform size and well dispersed.The average particle size was (57.66±7.30) nm calculated by Image J software.Compared with control group, the cell viabilities in SiNPs exposure groups were decreased(P<0.05), while the intracellular silicon levels and LDH activities in medium were increased (P<0.05);the ROS levels were increased, the MMP was declined, and the apoptosis appeared.All the cell effects mentioned above were enlarged in a dose-dependent manner. Conclusion SiNPs have toxicities to vascular endothelial cells, which could induce ROS generation and oxidative stress, further can do damage to cellular membrane and mitochondria, and thus lead to apoptosis eventually.

Key words: silica nanoparticle, cytotoxicity, apoptosis, mitochondrion

中图分类号:

R994

夏银叶, 李艳博, 牛丕业, 王晖, 王翼飞, 梁宝璐, 赵峰, 郭彩霞, 孙志伟. 纳米二氧化硅颗粒对血管内皮细胞的毒性及其凋亡诱导作用[J]. 吉林大学学报(医学版), 2015, 41(03): 454-459.

XIA Yinye, LI Yanbo, NIU Piye, WANG Hui, WANG Yifei, LIANG Baolu, ZHAO Feng, GUO Caixia, SUN Zhiwei. Toxicity of silica nanoparticles on vascular endothelial cells and its induction effect on apoptosis[J]. Journal of Jilin University Medicine Edition, 2015, 41(03): 454-459.

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纳米二氧化硅颗粒对血管内皮细胞的毒性及其凋亡诱导作用

Toxicity of silica nanoparticles on vascular endothelial cells and its induction effect on apoptosis

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