Name: rapid evaluation of toxicity of chemical compounds using zebrafish embryos
Uploaded: 2019-08-25T14:00:00
Description: Watch this Scientific Journal Video about Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos at JoVE.com

这项工作得到了西格丽德·朱塞柳斯基金会（SP，MP）、芬兰文化基金会（AA、MH）、芬兰学院（SP、MP）、奥里翁·法莫斯基金会（MH）、坦佩雷结核病基金会（SP、MH和MP）以及简和阿托斯·埃尔科基金会（SP和MP）的赠款支持。).我们感谢我们的意大利和法国合作者苏普兰教授和Winum教授为抗结核和抗癌药物开发目的提供安全和毒性评估的碳氢化物抑制剂。我们感谢奥里基·莱赫努斯和玛丽安·库斯拉赫蒂的技术援助。我们还感谢利娜·梅基宁和汉娜莱娜·皮波在斑马鱼繁殖和胚胎收集方面的帮助。我们衷心感谢哈兰·巴克对手稿的批判性评价和有见地的评论。

坦佩雷大学的斑马鱼核心设施拥有国家动物实验委员会（ESAVI/7975/04.10.05/2016）颁发的机构授权。所有使用斑马鱼胚胎的实验都按照东芬兰省政府、坦佩雷地区服务部社会和卫生部协议——LSLH-2007-7254/Ym-23进行。
1. 设置夜间斑马鱼交配罐
<ol><li>将2-5只成年雄性斑马鱼和3-5只成年雌性斑马鱼放入交配池过夜。（在早晨由自动黑暗和光循环诱导过夜）。</li>
	<li>设置几个十字架，?...

<ol>
	<li>Amaouche, N., Casaert Salome, H., Collignon, O., Santos, M. R., Ziogas, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marketing+authorization+applications+submitted+to+the+European+Medicines+Agency+by+small+and+medium-sized+enterprises:+an+analysis+of+major+objections+and+their+impact+on+outcomes.">Marketing authorization applications submitted to the European Medicines Agency by small and medium-sized enterprises: an analysis of major objections and their impact on outcomes.</a> Drug Discovery Today. 23 (10), 1801-1805 (2018).</li><li>Garg, R. C., Bracken, W. M., Hoberman, A. M., Gupta, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproductive+and+developmental+safety+evaluation+of+new+pharmaceutical+compounds.">Reproductive and developmental safety evaluation of new pharmaceutical compounds.</a> Reproductive and Developmental Toxicology. , 89-109 (2011).</li><li>Lee, H. Y., Inselman, A. L., Kanungo, J., Hansen, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+models+in+developmental+toxicology.">Alternative models in developmental toxicology.</a> Systems Biology in Reproductive Medicine. 58 (1), 10-22 (2012).</li><li>Gao, G., Chen, L., Huang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-cancer+drug+discovery:+update+and+comparisons+in+yeast,+Drosophila,+and+zebrafish.">Anti-cancer drug discovery: update and comparisons in yeast, Drosophila, and zebrafish.</a> Current Molecular Pharmacology. 7 (1), 44-51 (2014).</li><li>Brown, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+test+chemicals+for+the+ECVAM+international+validation+study+on+in+vitro+embryotoxicity+tests.+European+Centre+for+the+Validation+of+Alternative+Methods.">Selection of test chemicals for the ECVAM international validation study on in vitro embryotoxicity tests. European Centre for the Validation of Alternative Methods.</a> Alternatives to Laboratory Animals. 30 (2), 177-198 (2002).</li><li>Selderslaghs, I. W., Van Rompay, A. R., De Coen, W., Witters, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+screening+assay+to+identify+teratogenic+and+embryotoxic+chemicals+using+the+zebrafish+embryo.">Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo.</a> Reproductive Toxicology. 28 (3), 308-320 (2009).</li><li>Brannen, K. C., Panzica-Kelly, J. M., Danberry, T. L., Augustine-Rauch, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+zebrafish+embryo+teratogenicity+assay+and+quantitative+prediction+model.">Development of a zebrafish embryo teratogenicity assay and quantitative prediction model.</a> Birth Defects Research Part B Developmental and Reproductive Toxicology. 89 (1), 66-77 (2010).</li><li>Hermsen, S. A., van den Brandhof, E. J., van der Ven, L. T., Piersma, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relative+embryotoxicity+of+two+classes+of+chemicals+in+a+modified+zebrafish+embryotoxicity+test+and+comparison+with+their+in+vivo+potencies.">Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies.</a> Toxicology in Vitro. 25 (3), 745-753 (2011).</li><li>Lessman, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+developing+zebrafish+(Danio+rerio):+a+vertebrate+model+for+high-throughput+screening+of+chemical+libraries.">The developing zebrafish (Danio rerio): a vertebrate model for high-throughput screening of chemical libraries.</a> Birth Defects Research. Part C, Embryo Today: Reviews. 93 (3), 268-280 (2011).</li><li>Lantz-McPeak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+assay+using+high+content+screening+of+zebrafish+embryos.">Developmental toxicity assay using high content screening of zebrafish embryos.</a> Journal of Applied Toxicology. 35 (3), 261-272 (2015).</li><li>Truong, L., Harper, S. L., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+embryotoxicity+using+the+zebrafish+model.">Evaluation of embryotoxicity using the zebrafish model.</a> Methods in Molecular Biology. 691, 271-279 (2011).</li><li>Rodrigues, G. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design,+synthesis,+and+evaluation+of+hydroxamic+acid+derivatives+as+promising+agents+for+the+management+of+Chagas+disease.">Design, synthesis, and evaluation of hydroxamic acid derivatives as promising agents for the management of Chagas disease.</a> Journal of Medicinal Chemistry. 57 (2), 298-308 (2014).</li><li>Kanungo, J., Cuevas, E., Ali, S. F., Paule, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+model+in+drug+safety+assessment.">Zebrafish model in drug safety assessment.</a> Current Pharmaceutical Design. 20 (34), 5416-5429 (2014).</li><li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development.">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (24), 12965-12969 (2000).</li><li>Stainier, D. Y., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+system+to+study+cardiovascular+development.">The zebrafish as a model system to study cardiovascular development.</a> Trends in Cardiovascular Medicine. 4 (5), 207-212 (1994).</li><li>Aspatwar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroimidazole-based+inhibitors+DTP338+and+DTP348+are+safe+for+zebrafish+embryos+and+efficiently+inhibit+the+activity+of+human+CA+IX+in+Xenopus+oocytes.">Nitroimidazole-based inhibitors DTP338 and DTP348 are safe for zebrafish embryos and efficiently inhibit the activity of human CA IX in Xenopus oocytes.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 33 (1), 1064-1073 (2018).</li><li>Rami, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-targeting+carbonic+anhydrase+IX+inhibitors+by+a+new+series+of+nitroimidazole-sulfonamides/sulfamides/sulfamates.">Hypoxia-targeting carbonic anhydrase IX inhibitors by a new series of nitroimidazole-sulfonamides/sulfamides/sulfamates.</a> Journal of Medicinal Chemistry. 56 (21), 8512-8520 (2013).</li><li>Spitsbergen, J. M., Kent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+state+of+the+art+of+the+zebrafish+model+for+toxicology+and+toxicologic+pathology+research--advantages+and+current+limitations.">The state of the art of the zebrafish model for toxicology and toxicologic pathology research--advantages and current limitations.</a> Toxicologic Pathology. , 62-87 (2003).</li><li>Teraoka, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+cytochrome+P450+1A+is+required+for+circulation+failure+and+edema+by+2,3,7,8-tetrachlorodibenzo-p-dioxin+in+zebrafish.">Induction of cytochrome P450 1A is required for circulation failure and edema by 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish.</a> Biochemical and Biophysical Research Communications. 304 (2), 223-228 (2003).</li><li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+drug+discovery+in+the+zebrafish.">In vivo drug discovery in the zebrafish.</a> Nature Reviews Drug Discovery. 4 (1), 35-44 (2005).</li><li>Hill, A. J., Teraoka, H., Heideman, W., Peterson, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+vertebrate+for+investigating+chemical+toxicity.">Zebrafish as a model vertebrate for investigating chemical toxicity.</a> Toxicological Sciences. 86 (1), 6-19 (2005).</li><li>Kari, G., Rodeck, U., Dicker, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+an+emerging+model+system+for+human+disease+and+drug+discovery.">Zebrafish: an emerging model system for human disease and drug discovery.</a> Clinical Pharmacology and Therapeutics. 82 (1), 70-80 (2007).</li><li>Gourmelon, A., Delrue, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+in+Support+of+Internationally+Harmonised+OECD+Test+Guidelines+for+Assessing+the+Safety+of+Chemicals.">Validation in Support of Internationally Harmonised OECD Test Guidelines for Assessing the Safety of Chemicals.</a> Advances in Experimental Medicine and Biology. 856, 9-32 (2016).</li><li>Aspatwar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-CA-specific+inhibitor+dithiocarbamate+Fc14-584B:+a+novel+antimycobacterial+agent+with+potential+to+treat+drug-resistant+tuberculosis.">beta-CA-specific inhibitor dithiocarbamate Fc14-584B: a novel antimycobacterial agent with potential to treat drug-resistant tuberculosis.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 32 (1), 832-840 (2017).</li><li>Kazokaite, J., Aspatwar, A., Kairys, V., Parkkila, S., Matulis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorinated+benzenesulfonamide+anticancer+inhibitors+of+carbonic+anhydrase+IX+exhibit+lower+toxic+effects+on+zebrafish+embryonic+development+than+ethoxzolamide.">Fluorinated benzenesulfonamide anticancer inhibitors of carbonic anhydrase IX exhibit lower toxic effects on zebrafish embryonic development than ethoxzolamide.</a> Drug and Chemical Toxicology. 40 (3), 309-319 (2017).</li><li>Howe, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+reference+genome+sequence+and+its+relationship+to+the+human+genome.">The zebrafish reference genome sequence and its relationship to the human genome.</a> Nature. 496 (7446), 498-503 (2013).</li><li>Granato, M., Nusslein-Volhard, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+genes+controlling+development.">Fishing for genes controlling development.</a> Current Opinion in Genetics &amp; Development. 6 (4), 461-468 (1996).</li><li>Bambino, K., Chu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+in+Toxicology+and+Environmental+Health.">Zebrafish in Toxicology and Environmental Health.</a> Current Topics in Developmental Biology. 124, 331-367 (2017).</li><li>Goldsmith, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+pharmacological+tool:+the+how,+why+and+when.">Zebrafish as a pharmacological tool: the how, why and when.</a> Current Opinion in Pharmacology. 4 (5), 504-512 (2004).</li></ol>

使用培养细胞进行体外毒性试验，可以检测细胞的生存和形态学研究，提供有关试验化合物引起的毒性的有限信息。利用斑马鱼胚胎对化合物进行毒性筛选的优点是，在相关模型生物体胚胎发育过程中，可快速检测整个动物的化学诱导型态变化。大约70%的蛋白质编码人类基因在斑马鱼基因组中具有正交对应物。控制信号转导和发育的遗传途径在人和斑马鱼26</...

药物开发是一个昂贵的过程。在单一化合物获得美国食品和药物管理局（FDA）和欧洲药品管理局（EMA）批准之前，对数千种化合物进行筛选，费用超过10亿美元。在临床前开发期间，大部分费用是动物试验2需要的。为了限制成本，药物开发领域的研究人员需要替代模型来对化合物进行安全筛选3。因此，在药物开发的早期阶段，使用一种能以合适的模型快速评估化合物的安全性和毒性的方法是非常有益的。有几个协议已经用于涉及动物和细胞培养模型的化合物的毒性筛选，但没有一个协议被验证，并共同使用4，5。使用斑马鱼的现有协议长度不同，个别研究人员根据其便利性要求6、7、8、9评估毒性。10,11,12.
近段时间，斑马鱼已成为评估胚胎发育6、7号过程中化合物毒性的便捷模型。斑马鱼在评价化合物13方面有许多内在优势。即使是大规模的实验也是可以的，因为斑马鱼雌性可以产下200-300个卵子，这些卵子在外发育迅速，不需要外部喂养长达一周，而且透明。化合物可以直接添加到水中，在那里它们可以（取决于化合物的性质）通过幼虫扩散，孵化后，通过幼虫的皮肤，刺和嘴。由于胚胎体积小，实验不需要大量的化合物14。开发斑马鱼胚胎表达实现正常发育结果所需的大部分蛋白质。因此，斑马鱼胚胎是一个敏感的模型，用于评估潜在的药物是否会干扰具有发育意义的蛋白质或信号分子的功能。斑马鱼的器官在2-5 dpf15之间发挥作用，在这个胚胎发育的敏感时期有毒的化合物会导致斑马鱼幼虫的表型缺陷。这些型状变化可以很容易地检测使用简单的显微镜没有侵入性技术11。斑马鱼胚胎被广泛用于毒理学研究，因为它们的生物复杂性比使用细胞培养模型16、17的体外药物筛选要复杂得多。 作为一种脊椎动物，斑马鱼的遗传和生理组成与人类相当，因此斑马鱼和人类之间的化合物毒性相似。20,21,因此，斑马鱼是药物发现早期评估化合物毒性和安全性的宝贵工具。
在本文中，我们详细介绍了单个研究人员使用1-5天后受精（dpf）斑马鱼胚胎评估碳氢合酶（CA）抑制剂化合物的安全性和毒性的方法。该协议涉及将斑马鱼胚胎暴露于不同浓度的化学抑制剂化合物中，并研究胚胎发育过程中的死亡率和样型变化。在接触化合物结束时，确定该化学品的LC50剂量。该方法允许个人对1-5种测试化合物进行有效筛选，并根据需要使用该方法的人的经验需要8-10小时（图1）。图2概述了评估化合物毒性所需的每个步骤。CA抑制剂的毒性评估需要8天，包括建立交配对（第1天）;从繁殖罐中收集胚胎，清洗并转移到28.5°C培养箱（第2天）;将胚胎分配到24孔板的井中，并加入稀释的CA抑制剂化合物（第3天）;幼虫的型板分析和成像（第4-8天），以及LC50剂量（第8天）的测定。 该方法快速高效，需要少量的化合物和实验室的基本设施。

<table border="0" cellpadding="0" cellspacing="0" style="width:972px;" width="972">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">24-well plates</td>
			<td style="width:185px;">Nunc</td>
			<td style="width:260px;">Thermo Scientific</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Balance (Weighing scale)</td>
			<td style="width:185px;">KERN</td>
			<td style="width:260px;">PLJ3000-2CM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Balance (Weighing scale)</td>
			<td style="width:185px;">Mettler Toledo</td>
			<td style="width:260px;">AB104-S/PH</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">CaCl2</td>
			<td style="width:185px;">JT.Baker</td>
			<td style="width:260px;">RS421910024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Disecting Probe</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td>17-467-604&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">DMSO</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">D4540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Falcon tubes 15 mL</td>
			<td style="width:185px;">Greiner bio-one</td>
			<td style="width:260px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">High molecular weight methylcellulose</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">M0262&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Incubator for zebrafish larvae</td>
			<td style="width:185px;">Termaks</td>
			<td style="width:260px;">B8000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">KCL</td>
			<td style="width:185px;">Merck</td>
			<td style="width:260px;">1.04936.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Methyl Blue</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">28983-56-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">MgSO4</td>
			<td style="width:185px;">Sigma Aldrich, Germany</td>
			<td style="width:260px;">M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Microcentrifuge tubes</td>
			<td style="width:185px;">Starlab</td>
			<td style="width:260px;">S1615-5500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">NaCl</td>
			<td style="width:185px;">VWR Chemicals</td>
			<td style="width:260px;">27810.295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Paraffin Histoplast IM</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">8331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Pasteur pipette&#160;</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:260px;">86.1171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Petri dish</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td>101R20&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Petri plates</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:260px;">82.1473</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Pipette (1 mL and 200 &#956;L)</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">4641230N, 4641210N&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Plates 24-Well</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:260px;">142485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Steriomicroscope/Camera</td>
			<td style="width:185px;">Zeiss</td>
			<td style="width:260px;">Stemi 2000-C/Axiocam 105 color</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Vials (1.5 mL)</td>
			<td style="width:185px;">Fisherbrand</td>
			<td style="width:260px;">11569914</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:342px;">Zebrafish AB strains</td>
			<td style="width:185px;">ZIRC&#160;</td>
			<td style="width:260px;">&#160; ZL1&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid evaluation of toxicity of chemical compounds using zebrafish embryos

斑马鱼是一种广泛使用的脊椎动物模型生物体，用于疾病和表型药物发现。斑马鱼产生许多后代，具有透明的胚胎和快速的外部发育。因此，斑马鱼胚胎也可用于快速评估珍贵和小批量药物的毒性评估。本文介绍了一种利用1-5天受精后胚胎有效筛选化合物毒性的方法。胚胎通过立体显微镜进行监测，以调查因接触不同浓度化合物而导致的型板状缺陷。也确定了化合物的半最大致命浓度（LC50）。本研究需要3-6毫克的抑制剂化合物，整个实验大约需要8-10小时，由一个人在实验室有基本设施完成。目前的规程适用于测试任何化合物，以识别该化合物在药物发现的早期阶段的不可容忍的毒性或脱靶效应，并检测细胞培养或其他动物模型中可能遗漏的细微毒性影响。该方法减少了药物开发的程序性延迟和成本。

毒性评价的关键部分是在一个实验中测试一种或多种化合物的不同浓度。在开始时，选择用于评估毒性的化合物，测试每种化合物的浓度数量，并据此制作一个图表（图3）。我们为每个化合物使用独特的颜色来组织样品（图3）。使用耐溶剂标记和在板的底部或侧面的标签是重要的，以避免以后的混合。 如果化合物诱导接触不...

Watch this Scientific Journal Video about Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos at JoVE.com

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

斑马鱼胚胎用于评估化合物的毒性。它们从外部发展，对化学品敏感，能够检测细微的型型变化。实验只需要少量的化合物，直接添加到含有胚胎的板中，使测试系统高效且经济高效。

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

毒试程序将由我实验室的高级研究员阿肖克·阿斯帕瓦尔博士进行论证。我们的协议识别目标外效应，并检测在发现的早期阶段，在细胞培养或其他动物模型中可能遗漏的化学品的微妙毒性效应。我们技术的主要优点是：快速高效，需要量很少的化学，以及实验室的基本设施。毒性筛查将有助于我们确定其对导致结核病的分枝杆菌效率的浓度，以及进一步临床前计算。这种方法利用对化学品的离目标效应的洞察，因此，这种方法可用于任何研究领域，其中化学品的毒性是最重要的。这种方法非常简单，任何人都可以进行，没有经验。没有经验的人应该仔细计划实验，只使用一种化合物。这种方法提供化学物质的毒性作用，其形式是发展中的斑马鱼胚胎的表型变化，因此对被测试的化合物的安全性。首先，将两到五只成年雄性斑马鱼和三到五只成年雌性斑马鱼放入交配池过夜。早晨，打开光线，因为繁殖是由自动黑暗和随后的光循环诱导的。为了避免处理对动物的压力，让动物休息两个星期，然后再使用相同的个体进行繁殖。第二天中午前，使用细网过滤器收集胚胎，并把它们转移到含有E3胚胎培养的培养皿上。将培养皿放在立体显微镜下，检查每批胚胎。识别不透明的外观，并将其移除为未受精或死亡的胚胎。将胚胎在28.5摄氏度的孵化器中过夜。第二天早上，在立体显微镜下检查胚胎，取出任何不健康或死亡的胚胎。小心地使用巴氏移液器将一个胚胎移植到24井板的每个井中。确保每个井包含足够的E3介质，以覆盖胚胎。拿出储存在冰箱4摄氏度的含有抑制剂化合物的小瓶。使用分析平衡来称量适当量的化合物，并为 E3 介质或其他适当溶剂中的每种化合物准备 250 微升 100 毫摩尔库存溶液。然后，使用E3介质根据15毫升离心管的毒性水平对库存溶液进行连续稀释。从每个包含一个 DPF 胚胎的井中，使用巴氏移液器和 1 毫升移液器一次去除 E3 水。立即将库存溶液中每个稀释剂的 1 毫升分配到 24 孔板的孔中，从较低位置开始，向更高浓度移动。对于控制组，加入 E3 水或其他相关溶剂。用化合物的名称和浓度标记 24 个井板，并在培养箱中将板在 28.5 摄氏度。注意通过密封24个井板的两侧，在培养箱中不会蒸发油井中化学物质的稀释剂。在接触化合物24小时后，使用巴氏移液器将暴露在含有3%高分子量甲基纤维素的小培养皿中的每一浓度化合物中的幼虫转移。用金属探头，侧身铺。将培养皿放在连接到相机的立体显微镜下。拍摄图像，每天将图像保存在单独的文件夹中，直到实验结束。每天在联机表或打印的纸张上输入表中的所有观测值。对于神经毒性化合物暴露，四到五个DPF幼虫可能表现出异常的游泳模式。在这种情况下，通过捕获短短的 30 秒到 1 分钟幼虫视频来记录这些变化。在接触化合物五天后，注意一半的胚胎死亡浓度，因为每种化学物质中的致命浓度为50。使用合适的程序，为所有浓度的胚胎死亡率构建一个曲线。在该协议中，毒性评估的关键部分是在一个实验中测试一种或多种化合物的不同浓度。对于在幼虫中诱发任何表型缺陷的化合物，在接触该化学品后一至五天内每24小时记录一次缺陷。与控制相比，用βCA抑制剂处理的胚胎浓度为250微摩尔和125微摩尔，存在各种表型缺陷。例如，即使在第3天，未包扎的胚胎，弯曲的身体结构，未使用的蛋黄囊和心上水肿，并在治疗后五天内在幼虫中缺乏卵石囊。在另一项研究中，用CA抑制剂治疗的胚胎显示没有卵石囊和游泳膀胱。实验确定了一种具有代表性的复合碳氢化物9，在500微摩尔的胚胎发育过程中，它诱导了最小或无表型变化。该化合物表现出高LC50浓度。然而，在与对照组的比较中，发现300微摩尔的同一化合物在接触五天后在幼虫中具有神经毒性和诱发性厌食症。良好的胚胎质量对于化学品的毒性筛选非常重要。为了获得优质的胚胎，使用幼鸽成年斑马鱼进行繁殖。通过进行相关的体外和体内实验，可以进一步描述安全产生的化合物。在抗管杆菌药物开发领域，该技术有助于在体内对斑马鱼胚胎中分枝杆菌的抑制进行进一步的实验。该协议涉及使用可能对人类有毒的化学品。参与实验的人在处理化学品时应小心。

Research

JoVE Journal

Medicine

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

使用实时活体成像在肿瘤纳米粒子吸收的评价

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

科研

医学

Accurate and Simple Evaluation of Vascular Anastomoses in Monochorionic Placenta using Colored Dye

在单绒毛膜胎盘血管吻合用有色染料的精确和简单的评价

The presence of placental vascular anastomoses is a conditio sine qua non for the development of twin-to-twin transfusion syndrome (TTTS) and twin anemia ...

Induction of Myocardial Infarction in Adult Zebrafish Using Cryoinjury

在成年斑马鱼心肌梗死的感应使用Cryoinjury

The mammalian heart is incapable of significant regeneration following an acute injury such as myocardial infarction1. By contrast, urodele amphibians and ...

The &alpha;-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

The ÃƒÅ½Ã‚Â±-test: Rapid Cell-free CD4 Enumeration Using Whole Saliva

测试：α-细胞快速免费的CD4计数，使用全唾液

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

对黑色素瘤修饰符一个斑马鱼原地肿瘤模型的筛选

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

化疗引起的血管毒性 - 实时<em&gt;在体内</em&gt;船舶减值成像

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS

在免疫抑制剂他克莫司对干血斑使用LC-MS / MS定量

The calcineurin inhibitor tacrolimus is the cornerstone of most immunosuppressive treatment protocols after solid organ transplantation in the United ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

弥漫性光学光谱治疗急性电离辐射引起的皮肤毒性的定量评价使用小鼠模型

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Solubility of Hydrophobic Compounds in Aqueous Solution Using Combinations of Self-assembling Peptide and Amino Acid

自组装肽与氨基酸结合的水溶液中疏水性化合物的溶解度

Self-assembling peptides (SAPs) are promising vehicles for the delivery of hydrophobic therapeutics for clinical applications; their amphipathic ...

Evaluation of Cerebral Blood Flow Autoregulation in the Rat Using Laser Doppler Flowmetry

使用激光多普勒流测量测量大鼠脑血流自动调节的评估

When investigating the body&#39;s mechanisms for regulating cerebral blood flow, a relative measurement of microcirculatory blood flow can be obtained ...