关闭
 
读者在线:用户名 密码
首页 期刊简介 投稿须知 期刊目录 专家风采 编委会 特邀顾问 联系我们 移动出版
  1. 1
  2. 2
  3. 3
  4. 4
  5. 5



刊物信息

期刊名称:药物分析杂志
主管单位:中国科学技术协会
主办单位:中国药学会
承办:中国食品药品检定研究院
主编:金少鸿
地址:北京天坛西里2号
邮政编码:100050
电话:010-67012819,67058427
电子邮箱:ywfx@nicpbp.org.cn
国际标准刊号:ISSN 0254-1793
国内统一刊号:CN 11-2224/R
邮发代号:2-237
 

访问统计
您是第  4 9 4 6 0 9 4 位浏览者
您当前的位置:首页 >> 正文

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(英文):
分类号:R917
出版年·卷·期(页码):2019,39 (12):2107-2116
DOI: 10.16155/j.0254-1793.2017.01.01
-----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、1 000 μg·mL-1,对应于HaCaT细胞组为1.75、3.5、7、14、28 μg·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。

-----英文摘要:---------------------------------------------------------------------------------------

Objective: To evaluate the toxic effect and its mechanisms of silver nanoparticles (AgNPs) at equivalent exposure concentration on three-dimensional (3D) epidermal model (HaCaT model without cuticle),compared with two-dimensional (2D) 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 (GSH-Px) 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-1a increased (2.97 times and 11.55 times compared to control,respectively),but the release of IL-6 and IL-8 were decreased. Conclusion: HaCaT model can simulate the morphological structure of wounded skin and objectively evaluate the local effects of wound dressings. It is a candidate alternative model for evaluating the skin toxicity risk of nanomaterials.

-----参考文献:---------------------------------------------------------------------------------------

[1] ABADI PGS,SHIRAZI FH,JOSHAGHANI M,et al.Ag+-promoted zinc oxide[Zn(O):Ag]:a novel structure for safe protection of human skin against UVA radiation[J].Toxicol Vitro,2018,50:318.DOI:10.1016/j.tiv.2018.02.016
[2] GEN? H,BARUTCA B,KOPARAL AT,et al.Biocompatibility of designed MicNo-ZnO particles:cytotoxicity,genotoxicity and phototoxicity in human skin keratinocyte cells[J].Toxicol Vitro,2018,47:238.DOI:10.1016/j.tiv.2017.12.004
[3] LU MM,BAI J,SHAO D,et al.Antibacterial and biodegradable tissue nano-adhesives for rapid wound closure[J].Int J Nanomedicine,2018,13:5849.DOI:10.2147/IJN.S177109
[4] ZHUO F,ABOUREHAB MAS,HUSSAIN Z.Hyaluronic acid decorated tacrolimus-loaded nanoparticles:efficient approach to maximize dermal targeting and anti-dermatitis efficacy[J].Carbohydr Polym,2018,197:478.DOI:10.1016/j.carbpol
[5] RóNAVáRI A,IGAZ N,GOPISETTY M,et al.Biosynthesized silver and gold nanoparticles are potent antimycotics against opportunistic pathogenic yeasts and dermatophytes[J].Int J Nanomedicine,2018,13:695.DOI:10.2147/IJN.S152010
[6] AKHTAR N,PATHAK K.Carbon nanotubes in the treatment of skin cancers:safety and toxicological aspects[J].Pharm Nanotechnol,2017,5(2):95
[7] SLIEM MA,KARAS RA,HARITH MA.A promising protected ascorbic acid-hydroxyapatite nanocomposite as a skin anti-ager:A detailed photo-and thermal stability study[J].J Photochem Photobiol B,2017,173:661.DOI:10.1016/j.jphotobiol.2017.07.004
[8] CHERNOUSIVA S,EPPLE M,Silver as antibacterial agent:ion,nanoparticle,and metal[J].AngewChemInt Ed Engl,2013,52(6):1636
[9] MADHUMATHI K,SUDHEESH KUMAR PT,ABHILASH S,et al.Development of novel chitin/nanosilver composite scaffolds for wound dressing applications[J].J Mater Sci Mater Med,2010,21(2):807
[10] ARCHANA D,SINGH BK,DUTTA J,et al.Chitosan-PVP-nanosilver oxide wound dressing:in vitro and in vivo evaluation[J].Int J Biol Macromol,2015,73:49.DOI:10.1016/j.ijbiomac.2014.10.055
[11] AVT,DINDA,AK,KOUL V.Evaluation of nano hydrogel composite based on gelatin/HA/CS suffused with asiatic acid/ZnO and CuO nanoparticles for second degree burns[J].Mater Sci Eng C Mater Biol Appl,2018,89:378.DOI:10.1016/j.msec.2018.03.034
[12] CALIENNI MN,TEMPRANA CF,PRIETO MJ,et al.Nano-formulation for topical treatment of precancerous lesions:skin penetration,in vitro,and in vivo toxicological evaluation[J].Drug Deliv Transl Res,2018,8(3):496
[13] WU PC,HSIAO HT,LIN YC,et al.The analgesia efficiency of ultrasmall magnetic iron oxide nanoparticles in mice chronic inflammatory pain model[J].Nanomedicine,2017,13(6):1975
[14] SIVAKUMAR AS,KRISHNARAJ C,SHEET S,et al.Interaction of silver and gold nanoparticles in mammalian cancer:as real topical bullet for wound healing-A comparative study[J].In Vitro Cell Dev Biol Anim,2017,53(7):632
[15] ZANETTE C,PELIN M,CROSERA M,et al.Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line[J].ToxicolIn Vitro,2011,25(5):1053
[16] ABRUZZO A,ARMENISE N,BIGUCCI F,et al.Surfactants from itaconic acid:Toxicity to HaCaT keratinocytes in vitro,miceller solubilization,and skin permeation enhancement of hydrocortisone[J].Int J Pharm,2017,524(1-2):9
[17] SZMYD R,GORALCZYK AG,SKALNIAK L,et al.Effect of silver nanoparticles on human primary keratinocytes[J].Biol Chem,2013,394(1):113
[18] BOONKAEW B,KEMPF M,KIMBLE R,et al.Cytotoxicity testing of silver-containing burn treatments using primary and immortal skin cells[J].Burns,2014,40(8):1562
[19] KORANI M,REZAYAT SM,GILANI K,et al.Acute and subchronic dermal toxicity of nanosilver in guinea pig[J].Int J Nanomed,2011,6:855.DOI:10.2147/IJN.S17065
[20] PORTES P,GRANDIDIER MH,COHEN C,et al.Refinement of the Episkin protocol for the assessment of acute skin irritation of chemicals:follow-up to the ECVAM prevalidation study[J].Toxicol In Vitro,2002,16(6):765
[21] ROGUET R,COHEN C,DOSSOU KG,et al.Episkin,a reconstituted human epidermis for assessing in vitro the irritancy of topically applied compounds[J].Toxicol Vitro,1994, 8(2):283
[22] KANDáROVá H,LIEBSCH M,GENSCHOW E,et al.Optimisation of the EpiDerm test protocol for the upcoming ECVAM validation study on in vitro skin irritation tests[J].ALTEX,2004,21(3):107
[23] CHATTERJEE A,BABU RJ,KLAUSNER M,et al.In vitro and in vivo comparison of dermal irritancy of jet fuel exposure using EpiDerm (EPI-200) cultured human skin and hairless rats[J].Toxicol Lett,2006,167(2):85
[24] KANDAROVA H,LIEBSCH M,SPIELMANN H,et al.Assessment of the human epidermis model SkinEthic RHE for in vitro skin corrosion testing of chemicals according to new OECD TG 431[J].Toxicol Vitro, 2006,20(5):547
[25] NETZLAFF F,LEHR CM,WERTZ PW,et al.The human epidermis models EpiSkin,SkinEthic and EpiDerm:an evaluation of morphology and their suitability for testing phototoxicity,irritancy,corrosivity,and substance transport[J].Eur J Pharm Biopharm,2005,60(2):167
[26] TORNIER C,AMSELLEM C,FRAISSINETTE ADEB,et al.Assessment of the optimized SkinEthic reconstructed human Epidermis (RHE) 42 bis skin irritation protocol over 39 test substances[J].Toxicol Vitro,2010,24(1):245
[27] KATOH M,HAMAJIMA F,OGASAWARA T,et al.Assessment of human epidermal model LabCyte EPI-MODEL for in vitro skin irritation testing according to European Centre for the validation of alternative methods (ECVAM)-validated protocol[J].J Toxicol Sci,2009,34(3):327
[28] HIKIMA T,KANEDA N,MATSUO,K,et al.Prediction of percutaneous absorption in human using three-dimensional human cultured epidermis LabCyte EPI-MODEL[J].Biol Pharm Bull,2012,35(3):362
[29] OECD Guideline for the Testing of Chemicals.No.439:In Vitro Skin Irritation:Reconstructed Human Epidermis Test Method.Organisation for Economic Cooperation and Development,Paris[EB/OL]. 2015. http://www.oecd.org/chemicalsafety/testing/oecdguidelinesforthetestingofchemicals.htm
[30] 卢永波,张荣芳,许珊珊,等。一种用于体外测试的中国汉族人表皮替代模型[A].广州:中国毒理学会第六届全国毒理学大会论文摘要,2013:1 LU YB,ZHANG RF,XU SS,et al.An Epidermal Replacement Model of Chinese Han People Used for in vitro Testing[A].Guangzhou:The Sixth National Toxicology Conference of the Chinese Society of Toxicology,2013:1
[31] 姜珊,陈亮,吴美玉,等。金核/银壳纳米棒在三维皮肤模型中的透皮行为和对组织活力的影响[J].药物分析杂志,2019,39(3):377 JIANG S,CHEN L,WU MY,et al.Transdermal behavior of gold/silver shell nanorods in three-dimentional epidermal model and it`s effect on tissue activity[J].Chin J Pharm Anal,2019,39(3):377
[32] BIANCO C,KEZIC S,CROSERA M,et al.In vitro percutaneous penetration and characterization of silver from silver-containing textiles[J].Int J Nanomed,2015,10:1899
[33] 刘洋,卢涛,周宙霖,等. HaCaT表皮模型作为皮肤刺激性体外替代实验的可行性研究[J].中国修复与重建外科杂志,2017,31(10):1262 LIU Y,LU T,ZHOU ZL,et al.Study on feasibility of HaCaT epidermal model as an alternative to skin irritation in vitro[J].Chin Reconstr Surg J,2017,31(10):1262

欢迎阅读《药物分析杂志》!您是该文第 201位读者!

药物分析杂志 © 2009
地址:北京天坛西里2号 邮政编码:100050; 电子邮件:ywfx@nicpbp.org.cn

本系统由北京博思汇文数字科技有限公司设计开发 技术服务电话:400-921-9838