成人斑马鱼损伤模型研究泼尼松对再生骨组织的影响
Summary
在这里, 我们描述了3成人斑马鱼损伤模型及其与免疫抑制药物治疗的联合使用。我们为再生组织的成像和在其中检测骨矿化提供指导。
Abstract
斑马鱼能够在截肢后再生各种器官, 包括附属物 (鳍)。这涉及到骨头的再生, 这重生在受伤后大约两周内。此外, 斑马鱼能够在颅骨钻孔引流后迅速愈合骨骼, 修复可以很容易地引入斑马鱼骨鳍射线的骨折。这些损伤分析是测试药物对快速形成骨的作用的可行实验范式。在这里, 我们描述了这3损伤模型的使用和他们联合使用与系统性糖皮质激素治疗, 这会发挥骨抑制和免疫抑制作用。我们提供了如何准备成人斑马鱼免疫抑制剂治疗的工作流程, 说明了如何执行翅片截肢、颅骨骨钻孔引流和翅片骨折, 并描述了糖皮质激素的使用如何影响骨形成成骨细胞和单细胞/巨细胞谱系作为先天免疫的一部分在骨组织。
Introduction
斑马鱼是研究脊椎动物发育和疾病的强大动物模型。这是由于他们是小动物, 繁殖得非常好, 并且他们的基因组是完全排序和服从操作1。其他优势包括在不同阶段执行持续实时成像的选项, 包括成人斑马鱼2的活体成像, 以及在斑马鱼幼虫3中执行高通量药物筛查的能力。此外, 斑马鱼在包括骨骼在内的各种器官和组织中具有很高的再生能力, 因而成为研究骨骼疾病和修复4、5的有用系统。
糖皮质激素引起的骨质疏松症是一种由糖皮质激素长期治疗导致的疾病, 例如哮喘或类风湿性关节炎等自身免疫疾病治疗过程。在大约30% 的糖皮质激素治疗患者中发展, 并代表一个主要的健康问题6;因此, 深入研究免疫抑制对骨组织的影响是非常重要的。近年来, 开发了各种斑马鱼模型来处理其发病机制。糖皮质激素介导的骨丢失已被诱导在斑马鱼幼虫, 例如, 导致反作用化合物的鉴定增加在药物屏幕7的骨量。此外, 在斑马鱼鳞片的体外和体内8,9, 糖皮质激素诱导的骨抑制作用被模仿。这些检测是非常令人信服的方法, 特别是当它涉及到识别新的免疫抑制剂和骨合成代谢药物。然而, 他们只是部分考虑到内骨骼, 并没有在再生环境中执行。因此, 他们不允许调查糖皮质激素介导的作用在成人, 再生骨形成的快速模式。
在这里, 我们提出了一个协议, 使研究人员能够研究糖皮质激素介导的对成人斑马鱼骨骼再生的影响。损伤模型包括斑马鱼尾鳍的部分截肢, 头骨的钻孔引流, 以及翅片射线骨折的创建 (图 1A-1C), 并结合糖皮质激素暴露通过孵化 (图1E).我们最近使用了本协议的一部分来描述暴露于泼尼松 (通常规定的皮质类固醇药物之一) 对成人斑马鱼再生鳍和颅骨骨10的后果。在斑马鱼中, 泼尼松在单核细胞/巨噬细胞谱系10中导致成骨细胞增殖减少、成骨细胞分化不完全和细胞凋亡快速诱导。在本议定书中, 我们还描述了如何将骨折引入单个骨翅片11, 因为这种方法在研究骨折修复过程中的糖皮质激素介导的影响时可能很有用。这里介绍的方法将有助于进一步解决在快速再生的骨骼中的糖皮质激素作用的基本机制, 也可能被用于在斑马鱼组织再生的背景下系统药物管理的其他设置。
Protocol
Representative Results
Discussion
斑马鱼在骨骼研究中被证明是有用的在许多方面。所选突变体模仿人类疾病的某些方面, 如成骨不全症或骨关节炎23、24、25、26、27和幼虫以及鳞片被用来在小分子筛中识别骨合成代谢化合物7,28,29。在临床…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项研究得到了德累斯顿再生疗法中心 (“斑马鱼作为一种模型来解开糖皮质激素引起的骨质流失的机制”) 的资助, 另外还获得了德意志科技 (Transregio 67, 项目387653785) 至 FK。我们非常感谢1月卡斯林和 Avinash Chekuru 为他们的指导和协助钻孔引流 calvariae 和骨折的骨鳍射线。实验由 KG 和 FK 设计、执行和分析。FK 写了手稿。我们还要感谢卡瑞安兰伯特, 妮可 Cudak, 和其他成员的钮和品牌实验室的技术援助和讨论。我们也感谢玛丽卡费舍尔和依奇塔·福勒·弗兰科娃米希林为优秀的鱼护理和亨利埃特瞪大钮和乔希为校对手稿。
Materials
| Prednisolone | Sigma-Aldrich | P6004 | |
| Dimethylsulfoxid (DMSO) | Sigma-Aldrich | D8418 | |
| Ethyl-3-aminobenzoate methanesulfonate (MS-222) | Sigma-Aldrich | A5040 | |
| Blunt forceps | Aesculap | BD027R | |
| Fine forceps | Dumont | 91150-20 | |
| Scalpel | Braun | 5518059 | |
| Agarose | Biozym | 840004 | |
| Injection needle (0.3×13 mm) | BD Beckton Dickinson | 30400 | |
| Micro drill | Cell Point Scientific | 67-1000 | distributed e.g. by Harvard Apparatus |
| Steel burrs (0.5 µm diameter) | Fine Science tools | 19007-05 | |
| Artemia ssp. | Sanders | 425GR | |
| Pasteur pipette (plastic, Pastette) | Alpha Labs | LW4111 | |
| Paraformaldehyde | Sigma-Aldrich | 158127 | |
| Alizarin red S powder | Sigma-Aldrich | A5533 | |
| Alcian blue 8 GX | Sigma-Aldrich | A5268 | |
| Calcein | Sigma-Aldrich | C0875 | |
| Trypsin | Sigma-Aldrich | T7409 | |
| Stereomicroscope | Leica | MZ16 FA | with QIMAGING RETIGA-SRV camera |
| Stereomicroscope | Olympus | MVX10 | with Olympus DP71 or DP80 camera |
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