利用斑马鱼胚胎对化合物毒性的快速评价
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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</…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 24-well plates | Nunc | Thermo Scientific | |
| Balance (Weighing scale) | KERN | PLJ3000-2CM | |
| Balance (Weighing scale) | Mettler Toledo | AB104-S/PH | |
| CaCl2 | JT.Baker | RS421910024 | |
| Disecting Probe | Thermo Scientific | 17-467-604 | |
| DMSO | Sigma Aldrich, Germany | D4540 | |
| Falcon tubes 15 mL | Greiner bio-one | 188271 | |
| High molecular weight methylcellulose | Sigma Aldrich, Germany | M0262 | |
| Incubator for zebrafish larvae | Termaks | B8000 | |
| KCL | Merck | 1.04936.0500 | |
| Methyl Blue | Sigma Aldrich, Germany | 28983-56-4 | |
| MgSO4 | Sigma Aldrich, Germany | M7506 | |
| Microcentrifuge tubes | Starlab | S1615-5500 | |
| NaCl | VWR Chemicals | 27810.295 | |
| Paraffin Histoplast IM | Thermo Scientific | 8331 | |
| Pasteur pipette | Sarstedt | 86.1171 | |
| Petri dish | Thermo Scientific | 101R20 | |
| Petri plates | Sarstedt | 82.1473 | |
| Pipette (1 mL and 200 μL) | Thermo Scientific | 4641230N, 4641210N | |
| Plates 24-Well | Thermo Scientific | 142485 | |
| Steriomicroscope/Camera | Zeiss | Stemi 2000-C/Axiocam 105 color | |
| Vials (1.5 mL) | Fisherbrand | 11569914 | |
| Zebrafish AB strains | ZIRC | ZL1 |
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