基于HaCaT三维表皮模型的纳米银毒性及其毒性机制研究  

Toxic responses and it’s mechanisms of silvernanoparticles on HaCaT three-dimensional epidermal model

作　　者：姜珊 魏利娜[1] 张艳云 吴美玉[4] 邵安良[1] 王璐 柳琳 刘颖[4] 谢黎明[4] 吴刚[2] 陈亮[1] 徐丽明[1,2] JIANG Shan;WEI Li-na;ZHANG Yan-yun;WU Mei-yu;SHAO An-liang;WANG Lu;LIU Lin;LIU Ying;XIE Li-ming;WU Gang;CHEN Liang;XU Li-ming(National Institutes for Food and Drug Control,Beijing 102629,China;Department of Preclinical Medicine and Forensic,Baotou Medical College,Baotou 014040,China;Guangdong Biocell Biotechnology Co.Ltd,Guangzhou 523808,China;National Center for Nanoscience and Technology,Beijing 100190,China)

机构地区：[1]中国食品药品检定研究院,北京102629 [2]包头医学院基础医学与法医学院,包头014040 [3]广东博溪生物科技有限公司,广州523808 [4]国家纳米科学中心,北京100190

出　　处：《药物分析杂志》2019年第12期2107-2116,共10页Chinese Journal of Pharmaceutical Analysis

基　　金：国家重点研发计划“纳米产业技术标准的共性关键科学问题研究”,课题3“纳米生物医学应用准入的性能评价标准与规范(2016YFA0200900,2016YFA0200903)

摘　　要：目的:应用三维(3D)表皮模型(HaCaT模型,无角质层),与二维(2D)HaCaT细胞对比,评价等效浓度下纳米银(AgNPs)对HaCaT模型的毒性效应,并考察其毒性机制。方法:选用同等浓度,即:单位细胞数AgNPs暴露浓度(HaCaT模型组为62.5、125、250、500、1 000μg·mL^-1,对应于HaCaT细胞组为1.75、3.5、7、14、28μg·mL^-1),分别暴露于HaCaT模型及单层HaCaT细胞24 h后,用噻唑盐比色法(MTT)和乳酸脱氢酶释放试验(LDH)测定AgNPs在HaCaT模型和HaCaT细胞中的毒性效应。取同等AgNPs浓度(单位细胞0.7 ng·mL^-1)暴露组,用流式细胞仪检测模型或细胞中活性氧(ROS)含量的变化;用相应试剂盒检测模型或细胞中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)的活性及丙二醛(MDA)的含量;用酶联免疫吸附试验(ELISA)检测模型或细胞培养液中的炎性因子IL-1α、TNF-α、IL-6和IL-8的含量。结果:随着AgNPs浓度的增加,MTT实验显示HaCaT模型相对组织活力下降至(91.16±3.59)%^(75.66±1.66)%,而HaCaT细胞活性下降至(77.42±5.17)%^(22.43±2.48)%,二者均呈剂量-效应关系;LDH实验显示HaCaT模型的LDH释放量从(0.17±1.12)%升高至(44.9±3.61)%,而HaCaT细胞的LDH释放量从(55.99±1.58)%升高至(68.68±1.42)%,二者均呈剂量-效应关系。AgNPs作用于HaCaT模型和细胞后,均引起SOD和GSH-px合成量下降,ROS的释放量及MDA升高(分别为对照组的3.7倍和11.5倍);抗氧化剂(NAC)能够不同程度地减少以上氧化应激因子的变化;引起IL-1α释放量显著升高(分别为对照组的2.97倍和11.55倍),IL-6和IL-8的释放量降低(分别为对照组的2.09倍和1.36倍、1.4倍和3.83倍)。结论:HaCaT模型较好地模拟了创伤皮肤的形态结构,能够客观地评价创伤皮肤外用敷料的局部作用,是评价纳米材料皮肤毒性风险的候选替代模型。Objective:To evaluate the toxic effect and its mechanisms of silver nanoparticles(AgNPs) at equivalent exposure concentration on three-dimensional(3 D) epidermal model(HaCaT model without cuticle),compared with two-dimensional(2 D) HaCaT cells. Methods:The equivalent concentration(HaCaT model group was set as 62.5,125,250,500,1 000 μg·mL^-1,corresponding to 1.75,3.5,7,14,28 μg·mL^-1 in HaCaT cell group) were exposed to HaCaT model and HaCaT cells for 24 hours,and the toxic responses were assessed by thiazolyl [3-4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-bromide] ratio(MTT) assay and lactate dehydrogenase(LDH) release assay;In the exposure group with the same AgNPs concentration(0.7 ng·mL^-1 per cell),equivalent concentration,the changes of reactive oxygen species(ROS) content in the model or cells were detected by flow cytometry. The activity of superoxide dismutase(SOD),glutathione peroxidase(GSHPx) and the content of Malondialdehyd(MDA) in the model or cells were detected with the corresponding kit. The levels of inflammatory cytokines IL-1α、TNF-α、IL-6 和 IL-8 in the model or cell culture solution were detected by ELISA. Results:With the increase of AgNPs exposure concentration,the MTT assay showed that the relative tissue viability of HaCaT model decreased from(91.16±3.59)% to(75.66±1.66)%,while the relative cell viability of HaCaT cells decreased from(77.42±5.17)% to(22.43±2.48)%,both of which showed a dose-dependent manner. The LDH assay showed that the LDH release in HaCaT model increased from(10.17±1.12)% to(44.9±3.61)%,while the LDH release in HaCaT cells increased from(55.99±1.58)% to(68.68±1.42)%,both of which showed a dose-dependent manner. After AgNPs exposed on HaCaT model(500 μg·mL^-1) and cells(14 μg·mL-1),the synthesis of SOD and GSH-px were decreased,while the release of ROS and MDA(3.7-times and 11.5-times compared to control,respectively) were increased;and antioxidant NAC can decrease the changes of these oxidative stress factors;and the release of IL-1 a increased(2.97

关 键 词：纳米银 表皮模型(HaCaT) 皮肤毒性 氧化应激 炎症反应 

分 类 号：R917[医药卫生—药物分析学]