基于斑马鱼的水生生态毒理学实验教学体系的构建
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摘要
在我国现有的生态毒理学教学体系中,相对于理论教学,其实验教学体系的构建和实践相对滞后,阻碍了相关领域的人才培养。斑马鱼是国际上广受瞩目的小型模式鱼,本文在目前已成功开发并应用于科研的一系列斑马鱼毒性测试策略和方法的基础上,初步构建较为完整的水生生态毒理学实验内容和案例。虽然仅基于斑马鱼这一实验物种,但包括了致死效应、致畸效应、神经毒性、内分泌干扰效应、免疫毒性、遗传毒性等,涵盖不同生物学层面的多个毒理学终点,有助于充分训练学生的实验技能,巩固所学的理论知识。此外,在学生初步掌握上述实验手段的基础上,设置综合性或者设计性实验,可进一步培养学生的创造性思维和科研能力。本文对于现有的生态毒理学实验教学有一定的参考和借鉴意义。
Up to now,the establishment and practice of experimental teaching system in the ecotoxicology is lagging behind when compared with the theoretical teaching. Over the past two decades,zebrafish has been recognized as a primary small model fish for ecotoxicology. Therefore,based on the current toxicity testing strategy and method in zebrafish,the aim of this paper is to preliminarily establish an experimental teaching system in the aquatic ecotoxicology. The contents include a set of tests on the lethal effect,teratogenic effect,neurotoxicity,endocrine disrupting effect,immunotoxicity,and genotoxicity,which covers multiple endpoints in different biological levels. This system will be helpful to the students for the practice of experimental skill,and the consolidation of theoretical knowledge. Moreover,the comprehensive and designed experiments are recommended in order to develop the ability of creative thinking and scientific research. This paper can provide some references for the reform of current experi-mental teaching system in ecotoxicology.
Up to now,the establishment and practice of experimental teaching system in the ecotoxicology is lagging behind when compared with the theoretical teaching. Over the past two decades,zebrafish has been recognized as a primary small model fish for ecotoxicology. Therefore,based on the current toxicity testing strategy and method in zebrafish,the aim of this paper is to preliminarily establish an experimental teaching system in the aquatic ecotoxicology. The contents include a set of tests on the lethal effect,teratogenic effect,neurotoxicity,endocrine disrupting effect,immunotoxicity,and genotoxicity,which covers multiple endpoints in different biological levels. This system will be helpful to the students for the practice of experimental skill,and the consolidation of theoretical knowledge. Moreover,the comprehensive and designed experiments are recommended in order to develop the ability of creative thinking and scientific research. This paper can provide some references for the reform of current experi-mental teaching system in ecotoxicology.
引文
[1]周启星,孔繁翔,朱琳.生态毒理学[M].北京:科学出版社,2004
[2]孟紫强.生态毒理学[M].北京:高教出版社,2009
[3]孔志明,杨柳燕,尹大强,等.现代环境生物学实验技术和方法[M].北京:中国环境科学出版社,2005
[4]付保荣.环境污染生态毒理与创新型综合设计实验教程[M].北京:中国环境出版社,2016
[5]宋志慧.水生生态毒理学实验[M].北京:化学工业出版社,2008
[6]Rennekamp A J,Peterson R T.15 years of zebrafish chemical screening[J].Current Opinion in Chemical Biology,2015,24(1):58-70
[7]Garcia G R,Noyes P D,Tanguay R L.Advancements in zebrafish applications for 21st Century toxicology[J].Pharmacology&Therapeutics,2016,161:11-21
[8]Truong L,Reif D M,St Mary L,et al.Multidimensional in vivo hazard assessment using zebrafish[J].Toxicological Sciences,2014,137(1):212-233
[9]王佳佳,徐超,屠云杰,等.斑马鱼及其胚胎在毒理学中的实验研究与应用进展[J].生态毒理学报,2007,2(2):123-135Wang J J,Xu C,Tu Y J,et al.Experimental research and application of zebrafish and embryos in toxicology[J].Asian Journal of Ecotoxicology,2007,2(2):123-135(in Chinese)
[10]Howe K,Clark M D,Torroja C F,et al.The zebrafish reference genome sequence and its relationship to the human genome[J].Nature,2013,496(7446):498-503
[11]Westerfield M.The Zebrafish Book:A Guide for the Laboratory Use of Zebrafish(Danio rerio)[M].Eugene:University of Oregon Press,2000
[12]国家环境保护局.GB/T 13267—91水质物质对淡水鱼(斑马鱼)急性毒性测定方法[S].北京:中国标准出版社,1991
[13]孔志明.环境毒理学[M].南京:南京大学出版社,2012
[14]朱琳,史淑洁.斑马鱼胚胎发育技术在毒性评价中的应用[J].应用生态学报,2002,13(2):252-254Zhu L,Shi S J.Utilization of embryo development technique of Brachydanio rerio to evaluating toxicity on various chemicals[J].Chinese Journal of Applied Ecology,2002,13(2):252-254(in Chinese)
[15]Ahmad F,Noldus L P,Tegelenbosch R A,et al.Zebrafish embryos and larvae in behavioural assays[J].Behaviour,2012,149(10-12):1241-1281
[16]Noyes P D,Haggard D E,Gonnerman G D,et al.Advanced morphological-behavioral test platform reveals neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants[J].Toxicological Sciences,2015,145(1):177-195
[17]Audira G,Sampurna B,Juniardi S,et al.A simple setup to perform 3D locomotion tracking in zebrafish by using a single camera[J].Inventions,2018,3(1):11
[18]Mac Phail R C,Brooks J,Hunter D L,et al.Locomotion in larval zebrafish:Influence of time of day,lighting and ethanol[J].Neurotoxicology,2009,30(1):52-58
[19]彭涛,王思思,任琳,等.磷酸三苯酯对斑马鱼早期生命阶段的神经毒性研究[J].生态毒理学报,2016,11(1):254-260Peng T,Wang S S,Ren L,et al.Neurotoxicity of triphenyl phosphate on the early life stages of zebrafish[J].Asian Journal of Ecotoxicology,2016,11(1):254-260(in Chinese)
[20]Van der Ven L T M,Holbech H,Fenske M,et al.Vitellogenin expression in zebrafish Danio rerio:Evaluation by histochemistry,immunohistochemistry,and in situ m RNA hybridisation[J].Aquatic Toxicology,2003,65(1):1-11
[21]Sun L W,Wen L L,Shao X L,et al.Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish(Danio rerio)[J].Chemosphere,2010,78(7):793-799
[22]李玄,尹大强,于振洋,等.环境污染物免疫毒性及其研究技术[J].生态毒理学报,2013,8(6):857-863Li X,Yin D Q,Yu Z Y,et al.Progress in the technology and research of the immunotoxicity of environmental pollutants[J].Asian Journal of Ecotoxicology,2013,8(6):857-863(in Chinese)
[23]Renshaw S A,Loynes C A,Trushell D M I,et al.A transgenic zebrafish model of neutrophilic inflammation[J].Blood,2006,108(13):3976-3978
[24]Sengupta S,Bisson W H,Mathew L K,et al.Alternate glucocorticoid receptor ligand binding structures influence outcomes in an in vivo tissue regeneration model[J].Comparative Biochemistry and Physiology C-Toxicology&Pharmacology,2012,156(2):121-129
[25]Mathew L K,Simonich M T,Tanguay R L.AHR-dependent misregulation of Wnt signaling disrupts tissue regeneration[J].Biochemical Pharmacology,2009,77(4):498-507
[26]Keightley M C,Wang C H,Pazhakh V,et al.Delineating the roles of neutrophils and macrophages in zebrafish regeneration models[J].International Journal of Biochemistry&Cell Biology,2014,56(2):92-106
[27]Rehberger K,Werner I,Hitzfeld B,et al.20 Years of fish immunotoxicology—What we know and where we are[J].Critical Reviews in Toxicology,2017,47(6):509-535
[28]National Research Council of the National Academies.Toxicity Testing in the 21st Century:A Vision and a Strategy[M].Washington,DC:The National Academies Press,2007
[2]孟紫强.生态毒理学[M].北京:高教出版社,2009
[3]孔志明,杨柳燕,尹大强,等.现代环境生物学实验技术和方法[M].北京:中国环境科学出版社,2005
[4]付保荣.环境污染生态毒理与创新型综合设计实验教程[M].北京:中国环境出版社,2016
[5]宋志慧.水生生态毒理学实验[M].北京:化学工业出版社,2008
[6]Rennekamp A J,Peterson R T.15 years of zebrafish chemical screening[J].Current Opinion in Chemical Biology,2015,24(1):58-70
[7]Garcia G R,Noyes P D,Tanguay R L.Advancements in zebrafish applications for 21st Century toxicology[J].Pharmacology&Therapeutics,2016,161:11-21
[8]Truong L,Reif D M,St Mary L,et al.Multidimensional in vivo hazard assessment using zebrafish[J].Toxicological Sciences,2014,137(1):212-233
[9]王佳佳,徐超,屠云杰,等.斑马鱼及其胚胎在毒理学中的实验研究与应用进展[J].生态毒理学报,2007,2(2):123-135Wang J J,Xu C,Tu Y J,et al.Experimental research and application of zebrafish and embryos in toxicology[J].Asian Journal of Ecotoxicology,2007,2(2):123-135(in Chinese)
[10]Howe K,Clark M D,Torroja C F,et al.The zebrafish reference genome sequence and its relationship to the human genome[J].Nature,2013,496(7446):498-503
[11]Westerfield M.The Zebrafish Book:A Guide for the Laboratory Use of Zebrafish(Danio rerio)[M].Eugene:University of Oregon Press,2000
[12]国家环境保护局.GB/T 13267—91水质物质对淡水鱼(斑马鱼)急性毒性测定方法[S].北京:中国标准出版社,1991
[13]孔志明.环境毒理学[M].南京:南京大学出版社,2012
[14]朱琳,史淑洁.斑马鱼胚胎发育技术在毒性评价中的应用[J].应用生态学报,2002,13(2):252-254Zhu L,Shi S J.Utilization of embryo development technique of Brachydanio rerio to evaluating toxicity on various chemicals[J].Chinese Journal of Applied Ecology,2002,13(2):252-254(in Chinese)
[15]Ahmad F,Noldus L P,Tegelenbosch R A,et al.Zebrafish embryos and larvae in behavioural assays[J].Behaviour,2012,149(10-12):1241-1281
[16]Noyes P D,Haggard D E,Gonnerman G D,et al.Advanced morphological-behavioral test platform reveals neurodevelopmental defects in embryonic zebrafish exposed to comprehensive suite of halogenated and organophosphate flame retardants[J].Toxicological Sciences,2015,145(1):177-195
[17]Audira G,Sampurna B,Juniardi S,et al.A simple setup to perform 3D locomotion tracking in zebrafish by using a single camera[J].Inventions,2018,3(1):11
[18]Mac Phail R C,Brooks J,Hunter D L,et al.Locomotion in larval zebrafish:Influence of time of day,lighting and ethanol[J].Neurotoxicology,2009,30(1):52-58
[19]彭涛,王思思,任琳,等.磷酸三苯酯对斑马鱼早期生命阶段的神经毒性研究[J].生态毒理学报,2016,11(1):254-260Peng T,Wang S S,Ren L,et al.Neurotoxicity of triphenyl phosphate on the early life stages of zebrafish[J].Asian Journal of Ecotoxicology,2016,11(1):254-260(in Chinese)
[20]Van der Ven L T M,Holbech H,Fenske M,et al.Vitellogenin expression in zebrafish Danio rerio:Evaluation by histochemistry,immunohistochemistry,and in situ m RNA hybridisation[J].Aquatic Toxicology,2003,65(1):1-11
[21]Sun L W,Wen L L,Shao X L,et al.Screening of chemicals with anti-estrogenic activity using in vitro and in vivo vitellogenin induction responses in zebrafish(Danio rerio)[J].Chemosphere,2010,78(7):793-799
[22]李玄,尹大强,于振洋,等.环境污染物免疫毒性及其研究技术[J].生态毒理学报,2013,8(6):857-863Li X,Yin D Q,Yu Z Y,et al.Progress in the technology and research of the immunotoxicity of environmental pollutants[J].Asian Journal of Ecotoxicology,2013,8(6):857-863(in Chinese)
[23]Renshaw S A,Loynes C A,Trushell D M I,et al.A transgenic zebrafish model of neutrophilic inflammation[J].Blood,2006,108(13):3976-3978
[24]Sengupta S,Bisson W H,Mathew L K,et al.Alternate glucocorticoid receptor ligand binding structures influence outcomes in an in vivo tissue regeneration model[J].Comparative Biochemistry and Physiology C-Toxicology&Pharmacology,2012,156(2):121-129
[25]Mathew L K,Simonich M T,Tanguay R L.AHR-dependent misregulation of Wnt signaling disrupts tissue regeneration[J].Biochemical Pharmacology,2009,77(4):498-507
[26]Keightley M C,Wang C H,Pazhakh V,et al.Delineating the roles of neutrophils and macrophages in zebrafish regeneration models[J].International Journal of Biochemistry&Cell Biology,2014,56(2):92-106
[27]Rehberger K,Werner I,Hitzfeld B,et al.20 Years of fish immunotoxicology—What we know and where we are[J].Critical Reviews in Toxicology,2017,47(6):509-535
[28]National Research Council of the National Academies.Toxicity Testing in the 21st Century:A Vision and a Strategy[M].Washington,DC:The National Academies Press,2007