217—225.[8] Rietschel E T,Brade H,Hols t O,et al.Baterial endotoxin:chemical cons titution,biol ogical recognition,hos t res pons e,and immunological detoxifica-tion.Current Topics in Microbiology and Immunology,1996,216:39—81.[9] Fennrich S,Wendel A,Hartung T.New application of the human whole bl ood pyrogen as say(PyroChec k).AL TEX,1999,16(3):146—149.[10] 胡可可,夏振民.药品注射剂的热原检查.中国海洋药物杂志,1991,4:26—29.[11] Taktak Y S,Selkirk S,Bristow A F,et al.As say of Pyrogens by Interleukin-6R elease from Monocytic Cell Lines.J Pharm Phar macol.1991,43:578—582[12] Eperon S,Groote D D,Werner-Fel mayer G,et al.Human monocytoid cell lines as indicators of endotoxin:co mparison with rabbit pyrogen and Limulusamoeboc yte lysate assay.J Immunol Methods,1997:207,135—145.[13] Hartung T,Cramer R,Wendel A.Devel opment of a pyrogen tes t based on rabbit blood.ALTEX,1998,Suppl ement.[14] Fennric h S,Fischer M,Hartung T,et al.Detection of endotoxins and other pyrogens using human whole bl ood.Dev Biol Stand,1999,101:131—139.[15] Hartung T,Fennrich S,Fischer M,et al.Development and evaluation of a pyrogen tes t based on human whole blood.AL TEX,1998,15(5):9—10.Another Novel Test for the Detection of Pyrogen in Order to Replace the Rabbit Pyrogen Test by Determination of the Release of TNF-αfrom THP-1LinesHUANG Qing-quan,LI Guan-min,XI Ting-fei,HE Zheng-ming(National Institut e for t he C ontr ol of Phar maceutical and Bi ologic al Products,Beijing 100050,C hina)A bstract: The aim of this study was to development an in vitro test for the detection of pyr ogen in order to replace the rabbit pyrogen test.Recently the THP-1monocytic cells was assessed for tumor necross factor(TNF)-αfor the probability of the novel test.When THP-1lines is incubated in the presence of reference standard endotoxin,they secrete“endoge-nous pyrogen”TNF-αdeter mined by ELISA after a fixed incubation time(6hours).These results sho w that the correla-tion coefficient is greater than or equal to0.980in the range of0.32E U mL-200EU mL between the TNF-αand the en-dotoxin.The same result has got from the other kind endotoxin with equal effect in the LAL TAL test.Therefore we think this novel test will ser ve as an aternative pyrogen test.Key words:Pyrogen;Pyrogen test;Cytokine;THP-1,TNF-α稀有鲫———一种新的鱼类实验动物曹文宣 王剑伟(中国科学院水生生物研究所,武汉 430072)关键词:稀有鲫,实验动物,鱼类 鱼类作为低等脊椎动物的代表和水生态系统的代表,具有繁育力强、体外受精、体外发育、胚体透明等特点,过去一直是胚胎学、遗传学、水毒理学、行为科学、环境科学等领域常用的材料。
自从Streisinger关于斑马鱼人工雌核发育及纯系建立的文章在Nature上发表后,以斑马鱼、青为代表的鱼类很快成为了实验动物的“新宠”,并在发育遗传学、环境科学领域的研究中扮演了越来越重要的角色。
我国从20世纪90年代开始先后进行了稀有鲫、剑尾鱼、红鲫等鱼类的实验动物化研究,并于2001年成立了中国实验动物学会水生实验动物专业委员会。
稀有鲫(G obioc ypris rarus)是我国特有的一种小型鲤科鱼类,分布于四川省汉源县、石棉县、双流县、都江堰市、彭州市等地。
从1990年开始,中国科学院水生生物研究所以将它作为新的实验动物为目的,先后对其分布区域与生活习性、形态与分类地位、繁殖、胚胎发育、胚后发育、生长、摄食、对生态因子的适应性、核型与同工酶、饲养方法、繁殖技术、麻醉方法、近交系培育等方面进行了系统的研究。
现在,我们已对其饲养管理进行了规范化,并获得了全兄妹近交20代的鱼,离培育成标准实验动物的目标已非常接近。
稀有鲫作为一种新的实验动物具有以下优点:(1)成体全长38—85mm,饲养方便;(2)在饲养条件下,孵出后3个月部分个体性腺成熟,4个月左右即可产卵繁殖;(3)稀有鲫在14℃—30℃间可自然产卵,在实验室控温条件下可以实现周年繁殖,一年中任何时候均可得到卵和苗,不受季节限制;(4)属于连续产卵类型的鱼类,同一尾鱼每隔4d左右产卵一次,每次数百粒卵。
短期内可获得同一亲本的大量后代;(5)卵粘性,卵膜径1.25—1.70mm,较斑马鱼、青卵大。
卵膜透明,可清楚地观察胚胎发育,也便于核移植等实验操作;(6)胚胎发育温度适应范围广,在13℃—30℃胚胎发育正常,可通过控制温度来控制发育速度;(7)对温度、二氧化碳、溶氧的耐受能力强。
迄今为止,有关稀有鲫的文献有51篇,除关于其生物学特点的研究论文18篇以外,其余33篇都是稀有鲫作为实验鱼应用的报道。
据不完全统计,国内已有十几个大学和科研单位使用稀有鲫开展科学研究,这些工作涉及鱼病学、遗传学、环境科学、胚胎学、生理生态学等领域。
在鱼病学领域,王铁辉等证实稀有鲫对草鱼出血病病毒异常敏感,可以将稀有鲫作为草鱼抗出血病病毒育种研究的模型。
由于采用稀有鲫作为研究材料,克服了采用草鱼时个体大、世代周期长、繁殖期短、饲养与取材难等困难,从而大为加速了草鱼抗出血病病毒育种研究的进程。
在环境科学领域,许多试验证实,稀有鲫对重金属、农药等化学品非常敏感,是进行化学品毒性测试和环境水样毒性试验的理想材料。
周永欣等建立了稀有鲫的5d和7d亚慢性毒性试验模型。
中国环境科学研究院不仅利用稀有鲫进行了鱼类毒性试验,而且将稀有鲫作为受试生物列入《国家环境保护局合格实验室准则(1996)》和《水和废水监测分析方法》(第四版)中。
在二英的生态效应和早期预警研究方面,水生生物研究所的研究表明,稀有鲫对2,3,7,8-TCDD极其敏感,低浓度下可致畸、致死、肝细胞受损。
低浓度的2,3,7,8-TCDD即可诱导EROD酶活性,且酶的活性与暴露浓度有极好的剂量-效应关系,证明EROD 生物测试法是一种快速、简单、灵敏的生物方法,可作为生物标志物评价鱼体不同发育阶段的毒性效应和生物积累。
目前,中国科学院水生生物研究所、中国科学院生态环境研究中心等正利用稀有鲫为试验材料在“评价内分泌干扰物长期低剂量暴露影响的鱼类实验模型”、“环境内分泌干扰物的生物筛选”等方面开展工作。
可以相信,稀有鲫将在我国的水毒理学研究与环境监测方面发挥越来越重要的作用。
在遗传学领域,已有研究者开展了染色体组操作、单性发育、转基因、克隆等方面的工作。
贾方均等进行了稀有鲫人工雌核发育、人工雄核发育的研究,建立了稀有鲫单性发育技术,为快速建立纯系提供了一条新途径。
钟家玉等用稀有鲫作材料,优化了鱼类电脉冲-精子介导转基因方法。
胡炜等通过异种核移植、连续核移植对鱼类供体体细胞核的再程序化及重构胚发育阻滞的机制等克隆研究中的基础问题进行了探讨。
由于稀有鲫具有繁殖力高、一年四季都可繁殖、且性成熟快等特点,因而可以作为转基因、克隆等遗传学研究的理想模式种。
Rare Minnoe:A New Laboratory Animal in ChinaCAO Wen-xuan,W ANG Jian-wei(Ins tiute of Hydrobiol ogy,The C hine se Academy of Sci enc e,Wuhan430072,China)A bstract: As representative member of lo w-class vertebrate and aquatic ecosystem,fishes have high breeding ability with eggs external tertilized and transpar ent embryos developing synchronously outside the mother.They are used in re-searches of embr yology,enetics,aquatic toxicology,behavioral science and environmental science.Ever since George us-ing experimental gynogenesis method was published in nature in1981,zebra fish,medaka and other fishes became favor-ite laboratory animals and played more and more important roles in the studies of genetics and envir onmental science.In 1990s,Chinese researchers began studying on rare minno w,swordtail,red crucian carp and other fishes as laboratory ani-mals;consequently,a professional subgroup of Chinese Association for Laboratory Animal Sciences named Society of Ex-perimental Aquatic Animal was set up in2001to promote researcher in this area.Rare minno w,G obiocypris rarus,is a small c yprinid fish.Distributing in Hanyuan,Shimian Shuangliu,Dqjiangy-an,Pengzhou County in Sichuan province,it is one of the endemic fishes in west China.In order to use rar e minnow as laboratory animal,scientists of Institute of Hydrobiology belonging to the Chinese Academy of Sciences(C AS)have done a series of investigation from1990.These works refer to its geographical distribution,habitat,morphology,taxonom y,re-production,ontogenesis,growth,food,semitiviqto ecological factors,karyotype,isozymes,breed and care,anaesthesia, pr oduction of inbred strain,and soon.Rare minnow has been inbred to20th generation at present,and guidelines for the laboratory culture and care of the fish have been made.This implies the time we produce and use rare minnow as stan-dard animal model is near.As a new laboratory animal,rare minnow has the flo wing attributes:(1)The ash is small with adult total length38to85mm,and it is easy to care for.(2)Some individuals get mature when they are3months old un-der favorite culture,and can spawn when they are4months old.The generation time is only4months.(3)Spa wning oc-curs when water temperature ranges fr om14to30degree of centigrade,thus it is easy to obtain eggs in my season in the laboratory.(4)Rare minnow is a continuous batch spawner.Mature females lay several hundr ed eggs at interval of a few days(mode4days).This ensures researchers can get large number of eggs spa wn by the same female in a shod period.(5)The eggs are adherent and transparent with diameter1.25to1.70mm,which are lar ger than eggs of zebra fish or medaka.E mbr yogenensis and organogenesis can be monitored microscopically,and it is feasible to transplant nuclear or othertechnical operate.(6)Development is nor mal between13and30degree of centigrade.It is possible to control the speed of ontogenesis by adjusting temperature.(7)The fish has high adaptability to temperature,high CO2and lo w dis-solved oxygen.There ar e51literatures on rare minnow presently.Eighteen of them ar e about biological background or taxonomy, and the other thirty-three ar e papers about using this fish as experimental animal.In complete statistic shows rare minnow has been used in studies on fish disease,genetics,environmental science,embryology,physiology and ecology in more than10universities or research institutions in China.Wang Tiehui et al proved that rar e minnow is susceptible to Hemorrhagic Virus of Grass Carp(GCHV)L,suggested the ash may serve as a model of the virus-resistance breeding of grass carp3with biotechnique ing rare min-now as experimental fish instead of grass carp,investigators overcomes the difficulties brought by big body size,long gen-eration time,short reproductive season,hard to care for and discommodious to obtain research materials,and hence it ac-celerates the course of the virus-resistance breeding.In environmental science research area,many experiments indicated that rare minnow is a suitable test organism for deter mining toxicity of chemicals and water sa mples because the fish is ver y sensitive to heavy metals,pesticides,or other che micals.Zhou Yongxin et al have established rare minnow5-day and7-day subchronic toxicity test models.Based on toxicity test data,scientists fr om Chinese Research Academ y of Envir onmental Sciences suggested rar e minno w as a rec-ommended test fish for acute toxicity test,and list it in Good Laboratory Practices(GLP)G uideline(National Environmental Protection Agenc y)and M ethod for examination of water and waste water(4th edi-tion).Researches on ecological effect and early warning system of dioxin indicated that rue minnow is susc eptivity to2, 3,7,8-TCDD.When exposed to low concentration of TCDD,abnormality,death,or damnification of liver cells was ob-served.During a longtime exposure at lo w dose,EROD activity be induced and a good dose-response relationship be-tween EROD activity and TCDD was obtained.This demonstrated ER OD is available as bioindicator in assessing the toxic effects of dioxins in fish during different development ing rare minnow as test animal,scientists from Institute ofHydrobidogy C AS and Research Center of Ec o·environmental Sciences CAS are studying on ec otoxicdogical effect of in-cr etion-interfere chemicals and risk assessment with long ter m exposure to low concentration.It is believed that rare min-now will act as a leading actor in aquatic toxicology and environment monitoring in China.In genetics research area,studies on genome manipulation,artificial unisexual development,gene transfer,nuclear transplantation were done in recent years.Jia Fangun et al explored the methods and tec hniques of artificial gynogenesis and androgenesis on rare minno w. This establishes a rapid approach of pr oduction homozygous diploid or pur eline.Zhong Jiayu developed and optimized a simple and faster gene transferring method,electr oporated-sperm-mediated gene transfer.It is proved that electroporation can force the foreign gene enter the sperm of rare minno w.Through nuclear transplantation between species and serial nu-clear transplantation of somatic cell,Hu Wei deduced a possible mechanism of somatic cell reprogra mming and develop-mental block of reconstructed embryos.Because of the idiosyncrasies such as high fecundity,laying eggs all the year round,getting mature rapidly,rare minnow is certainly a good and available model fish for gene transfer,clone,and oth-er genetic researches.Key words:Rare minnow;Laborator y animal;Fish实验动物饲养管理中的动物福利徐 平(中国科学院上海实验动物中心,上海 201615)关键词:实验动物;动物福利动物福利,其定义为“动物与它的环境相协调一致的精神和生理完全健康的状态。