斑马鱼细胞系的细胞毒性测定
Summary
该协议介绍了常用的细胞毒性测定（阿拉马尔蓝 [AB]、CFDA-AM、中性红和 MTT 测定），适用于评估斑马鱼胚胎 （ZEM2S） 和肝脏 （ZFL） 细胞系的细胞毒性 96 孔板。
Abstract
鱼细胞系越来越多地用于生态毒性研究，细胞毒性测定已被提出作为预测鱼类急性毒性的方法。因此，该协议提出了修改的细胞毒性测定，以评估斑马鱼（Danio rerio）胚胎（ZEM2S）和肝脏（ZFL）细胞系中的细胞活力，该板在96孔板中。评估的细胞毒性终点是线粒体完整性（Alamar Blue [AB] 和 MTT 测定）、通过酯酶活性 的 膜完整性（CFDA-AM 测定）和溶酶体膜完整性（中性红 [NR] 测定）。在96孔板中暴露测试物质后，进行细胞毒性测定;在这里，AB和CFDA-AM同时进行，然后在同一板上进行NR，而MTT测定在单独的板上进行。这些测定的读数通过AB和CFDA-AM的荧光以及MTT和NR的吸光度获取。用这些鱼细胞系进行的细胞毒性测定可用于研究化学物质对鱼的急性毒性。
Introduction
需要对化学物质进行人体健康和环境安全性测试。监管机构和/或立法（例如，REACH、OECD、US EPA）在安全性评估中越来越多地考虑分子和细胞生物标志物来预测对生物体的影响1，2，因为它们可以先于体内不良结果（例如，内分泌干扰、免疫反应、急性毒性、光毒性）3，4，5，6，7.在这种情况下，细胞毒性已被作为预测鱼类急性毒性的测量方法5，8;然而，它可以在生态毒性研究中有许多其他应用，例如定义化学物质的亚细胞毒性浓度，以研究它们对鱼类最多样化的影响（例如，内分泌干扰效应）。
在细胞培养系统（体外 系统）中，化学物质的细胞毒性可以通过终点类型的不同方法确定。例如，一种细胞毒性方法可以基于与细胞死亡过程中观察到的特定形态相关的终点，而另一种方法可以通过测量细胞死亡、活力和功能、形态、能量代谢以及细胞附着和增殖来确定细胞毒性。化学物质可以通过不同的机制影响细胞活力，因此覆盖不同细胞活力终点的细胞毒性评估对于预测化学效应是必要的9。
MTT和Alamar Blue（AB）是根据细胞代谢活性确定对细胞活力影响的测定。MTT测定评估线粒体酶琥珀酸脱氢酶10的活性。淡黄色的3-[4，5-二甲基噻唑-2基]-2，5-二苯基四唑溴化物（MTT）还原为甲臜蓝仅发生在活细胞中，其光密度与活细胞的数量成正比10。AB测定是一种灵敏的氧化还原指示剂，由线粒体酶介导，线粒体酶在活细胞将刃天青还原为试卤灵时发出荧光并改变颜色11;然而，胞质和微粒体酶也有助于减少AB和MTT12。这些酶可能包括几种还原酶，例如醇和醛氧化还原酶、NAD（P）H：醌氧化还原酶、黄素还原酶、NADH脱氢酶和细胞色素11。
中性红（NR）测定是基于将该染料掺入活细胞的溶酶体中的细胞活力测定13。NR的摄取取决于细胞维持pH梯度的能力。溶酶体内的质子梯度保持低于细胞质的pH值。在正常的生理pH值下，NR呈现的净电荷约为零，这使其能够穿透细胞膜。因此，染料带电并保留在溶酶体内。因此，保留NR的量越大，活细胞的数量就越多14。破坏细胞表面或溶酶体膜的化学物质会损害这种染料的摄取。
CFDA-AM测定是基于保留5-羧基荧光素二乙酸乙酰氧基甲酯（CFDA-AM）的荧光细胞活力测定15。5-CFDA-AM是一种酯酶底物，被转化为羧基荧光素，羧基荧光素是一种极性且不可被活细胞膜渗透的荧光物质15;因此，它保留在完整细胞膜的内侧，表明活细胞。
最近，三种细胞毒性测定（CFDA-AM、NR 和 AB 测定）在经过验证的 ISO（国际标准化组织）指南 （ISO 21115：2019）16 和 OECD（经济合作与发展组织）测试方法 （OECD TG 249） 中合并，以使用 RTgill-W1 细胞系（虹鳟鱼 [Oncorhynchus mykiss] 鳃的永久性细胞系）在 24 孔板中评估鱼类急性毒性17.虽然有一种现有的基于细胞的方法来预测鱼类的急性毒性，但已经投入了努力开发与其他鱼类物种类似的方法，并提高该方法的通量。一些例子包括用特定毒性途径18，19的报告基因转染的ZFL细胞系的开发，RTgill-W1细胞系20中的光毒性测试，以及使用ZFL和ZF4细胞系（来自1天龄胚胎的斑马鱼成纤维细胞）通过几种细胞毒性测定来评估毒性21。
Danio rerio（斑马鱼）是水生毒性研究中使用的主要鱼类之一;因此，使用斑马鱼细胞系进行基于细胞的鱼类毒性测试方法可能非常有用。ZFL细胞系是一种斑马鱼上皮肝细胞系，具有肝实质细胞的主要特征，可代谢异生素7，22，23，24，25。同时，ZEM2S细胞系是源自胚泡阶段的胚胎斑马鱼成纤维细胞系，可用于研究对鱼类26，27的发育影响。因此，该协议描述了四种细胞毒性测定（MTT，AB，NR和CFDA-AM测定），并在96孔板中使用ZFL和ZEM2S细胞系进行修饰。
Protocol
注意：有关本协议中使用的材料列表，请参阅 材料 表，有关本协议中使用的溶液和介质的组成，请参阅 表1 。 1. 制备ZFL和ZEM2S细胞 从具有80%汇合度的ZFL或ZEM2S细胞的T75烧瓶开始，在28°C的相应完全培养基中培养，不含CO2。 从烧瓶中取出培养基，并通过加入 10 mL 1x 磷酸盐缓冲盐水 （PBS） （0.01 M） 洗涤细胞。向培?…
Representative Results
图3显示了AB，CFDA-AM，NR和MTT测定的板。对于AB测定（图3A），空白孔和没有或减少活细胞数量的孔显示蓝色和低荧光，而具有大量活细胞的孔呈粉红色并呈现高荧光值，因为刃天青（AB）转化为试卤灵（粉红色物质）被活细胞。对于CFDA-AM测定，板上孔的颜色没有明显差异;然而，由于CFDA-AM的保留并随后转化为羧基荧光素（荧光物质），含有活细胞的孔中?…
Discussion
细胞毒性测定广泛用于体外毒性评估，本协议文章介绍了四种常用的细胞毒性测定，修改后可在斑马鱼细胞系中进行（即，96孔板的细胞密度，MTT测定中的孵育时间，化学暴露条件下的FBS耗竭和SC的最大可接受浓度）。由于这些测定通过不同的细胞活力终点（代谢功能、溶酶体膜完整性和细胞膜完整性）量化细胞毒性，因此它们的组合提供了斑马鱼细胞系中化学细胞毒性的准确评估。该协议…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
为了纪念这部作品的合著者Márcio Lorencini博士，他是化妆品领域的优秀研究人员，致力于促进巴西的化妆品研究。作者感谢生理学系（UFPR）的多用户实验室提供设备，并感谢高等教育人员改进协调（CAPES，巴西）（财务代码001）和Grupo Boticario的财政支持。
Materials
| 5-CFDA, AM (5-Carboxyfluorescein Diacetate, Acetoxymethyl Ester) | Invitrogen | C1345 | |
| Cell culture plate, 96 well plate | Sarstedt | 83.3924 | Surface: Standard, flat base |
| DMEM | Gibco | 12800-017 | Powder, high glucose, pyruvate |
| Ham's F-12 Nutrient Mix, powder | Gibco | 21700026 | Powder |
| HEPES (1 M) | Gibco | 15630080 | |
| Leibovitz's L-15 Medium | Gibco | 41300021 | Powder |
| Neutral red | Sigma-Aldrich | N4638 | Powder, BioReagent, suitable for cell culture |
| Orbital shaker | Warmnest | KLD-350-BI | 22 mm rotation diameter |
| Dulbeccos PBS (10X) with calcium and magnesium | Invitrogen | 14080055 | |
| Penicillin-Streptomycin (10,000 U/mL) | Gibco | 15140122 | |
| Resazurin sodium salt | Sigma-Aldrich | R7017 | Powder, BioReagent, suitable for cell culture |
| RPMI 1640 Medium | Gibco | 31800-014 | Powder |
| SFB – Fetal Bovine Serum, qualified, USDA-approved regions | Gibco | 12657-029 | |
| Sodium bicarbonate | Sigma-Aldrich | S5761 | Powder, bioreagent for molecular biology |
| Thiazolyl Blue Tetrazolium Bromide 98% | Sigma-Aldrich | M2128 | |
| Trypan blue stain (0.4%) | Gibco | 15250-061 | |
| Trypsin-EDTA (0.5%), no phenol red | Gibco | 15400054 | |
| ZEM2S cell line | ATCC | CRL-2147 | This cell line was kindly donated by Professor Dr. Michael J. Carvan (University of Wisconsin, Milwaukee, USA) |
| ZFL cell line | BCRJ | 256 |
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Citazione di questo articolo
Rodrigues de Souza, I., Wilke Sivek, T., Vaz de Oliveira, J. B., Di Pietro Micali Canavez, A., de Albuquerque Vita, N., Cigaran Schuck, D., Rodrigues de Souza, I., Cestari, M. M., Lorencini, M., Leme, D. M. Cytotoxicity Assays with Zebrafish Cell Lines. J. Vis. Exp. (191), e64860, doi:10.3791/64860 (2023).