使用茜素红染色检测斑马鱼幼虫中化学诱导的骨质流失
1Department of Toxicology, School of Public Health, Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases,Medical College of Soochow University, 2Department of Occupational Disease Prevention,Jiangsu Provincial Center for Disease Control and Prevention, 3Jiangsu Preventive Medicine Association, Nanjing, China
Summary
在这里，我们使用茜素红染色来证明醋酸铅暴露会导致斑马鱼幼虫的骨量变化。这种染色方法可以适用于研究由其他有害毒物引起的斑马鱼幼虫损失的骨质流失。
Abstract
铅（Pb）暴露引起的化学诱导骨质流失可能会对人类和动物骨骼系统产生一系列不利影响。然而，斑马鱼的具体效果和机制尚不清楚。茜素红对钙离子具有很高的亲和力，可以帮助可视化骨骼并说明骨骼矿物质质量。在这项研究中，我们旨在通过使用茜素红染色来检测醋酸铅（PbAc）诱导的斑马鱼幼虫骨质流失。在受精后2至120小时之间用一系列PbAc浓度（0，5，10，20mg / L）处理斑马鱼胚胎。在受精后9天对幼虫进行全支架骨骼染色，并使用ImageJ软件定量总染色面积。结果表明：PbAc暴露组矿化组织呈红色，染色面积显著减少，骨矿化度呈剂量依赖性变化。本文提出了一种染色方案，用于研究PbAc诱导的骨缺损的骨骼变化。该方法还可用于斑马鱼幼虫，以检测由其他化学物质引起的骨质流失。
Introduction
最近的研究证实，糖皮质激素、芳香酶抑制剂和过量饮酒引起的骨质疏松症很常见1，2.铅 （Pb） 是一种有毒金属，存在于植物、土壤和水生环境中3.虽然Pb对人体的不良影响备受关注，但其对骨骼的不可逆影响仍需进一步研究。铅中毒会导致发育和成人骨骼发生多种病理变化，影响正常生活活动。研究发现，慢性铅暴露与骨损伤之间存在关联4，包括骨骼结构受损5，6，骨矿物质密度降低，甚至骨质疏松症风险增加7。
矿化组织对骨强度非常重要8，骨矿化基质沉积是骨形成的关键指标9。茜素红对钙离子具有很高的亲和力，茜素红染色是评估骨形成的标准程序10。根据这种方法，矿化组织被染成红色，而所有其他组织保持透明。然后通过数字图像分析11对染色区域进行量化。
斑马鱼是一种重要的模式生物，广泛用于药物发现和疾病模型。斑马鱼和人类的遗传研究表明，在分子水平上骨骼形态发生的潜在机制相似12。此外，与鼠模型相比，高通量药物或生物分子筛选在大离合斑马鱼中更可行，促进了成骨或骨毒性分子的机理研究13。 toto10 中骨骼的差异染色经常用于研究小型脊椎动物和哺乳动物胎儿的骨骼发育不良。采用茜素红染色法研究斑马鱼幼虫中化学物质的骨发育毒性。本文以铅为例描述了检测斑马鱼幼虫中醋酸铅诱导的骨缺陷的方案。
Protocol
此处概述的所有动物程序均已由苏州大学伦理委员会动物护理研究所审查和批准。 1. 鱼类饲养与胚胎采集 14 每天喂鱼三次;确保斑马鱼保持在28.5±0.5°C，光照/黑暗循环为14：10小时。 晚上用产卵池中的隔离板将雄鱼和雌鱼分开，男女比例为2：1。 第二天早上，在上午9：00移除隔离板，并在2小时后收集胚胎。 在实验?…
Representative Results
茜素红染色是测量斑马鱼幼虫骨矿化变化的灵敏特异方法。在这项研究中，我们观察到PbAc对斑马鱼幼虫有不利影响，包括死亡、畸形、心率下降和体长缩短。此外，还评估了斑马鱼幼虫的矿物骨骼区域，以检查PbAc诱导的骨质流失。在9 dpf（图1A）时，头部骨骼的许多骨骼被矿化，因此染成红色，例如蝶旁（PS），眼睑（OP），角分支（CB）和脊索（NC）。相比之下，耳石（OT）…
Discussion
斑马鱼是研究骨代谢疾病的合适模型。与啮齿动物模型相比，斑马鱼模型的建立速度相对较快，并且更容易测量疾病的严重程度。在野生型斑马鱼幼虫中，头部骨骼的矿化发生在5 dpf，轴骨架的矿化发生在7 dpf15。因此，颅骨如PS，OP，CB和NC在9 dpf时发育良好。幼虫完全脱色漂白后，清除软组织，使鱼体外观透明。加入茜素红染色试剂，对斑马鱼的矿骨进行染色和可视化。
<p cla…Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（81872646; 81811540034; 81573173）和江苏省高等教育机构优先学术项目开发（PAPD）的支持。
Materials
| 1 M Tris-HCl (pH=7.5) | Solarbio,Beijing,China | 21 | for detaining |
| 4% Paraformaldehyde Fix Solution | BBI,Shanghai,China | 14 | fixing tissues |
| 10x PBS buffer | BBI,Shanghai,China | 15 | for fixing |
| 35% H2O2 | Yonghua,Jiangsu,China | 8 | removing pigment |
| 50 mL Centrifuge tube | AKX,Jiangsu,China | 4 | |
| 95% Anhydrous ethanol | Enox,Jiangsu,China | 2 | destaining |
| Alizarin red (Purity 99.5%) | Solarbio,Beijing,China | 1 | staining |
| Biochemical incubator | Yiheng,Shanghai,China | 3 | raising zebrafish embryos |
| Electronic scale | Sartorius,Germany | 5 | weighing the solid raw materials |
| Glycerin (Purity 99.5%) | BBI,Shanghai,China | 7 | storing the stained fish |
| ImageJ (software) | USA | 9 | digital analysis |
| KOH (Purity 99.9%) | Sigma,America | 10 | bleaching solution |
| Lead acetate trihydrate (Purity 99.5%) | Aladdin,Shanghai,China | 11 | |
| MgCl2 (Purity 99.9%) | Aladdin,Shanghai,China | 12 | cleaning solution |
| NIS-Elements F (software) | Nikon, Japan | 13 | observing and taking photos |
| Pipe | AKX, Jiangsu, China | 18 | removal of embryos and solution |
| plates (24-well) | Corning,America | 17 | container for staining embryos |
| plates (6-well) | Corning,America | 16 | container for breeding embryos |
| Shaking table | Beyotime, China | 19 | mixing the solution |
| Stereo microscope | Nikon,Japan | 20 | observing and taking photos |
| Zebrafish | Zebrafish Experiment Center of Soochow University,Suzhou,China | 22 | experimental animal |
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Diesen Artikel zitieren
Ding, J., Yan, R., Wang, L., Yang, Q., Zhang, X., Jing, N., Wei, Y., Zhang, H., An, Y. Using Alizarin Red Staining to Detect Chemically Induced Bone Loss in Zebrafish Larvae. J. Vis. Exp. (178), e63251, doi:10.3791/63251 (2021).