阿霉素诱发成年斑马鱼的心肌病模型
Summary
本文介绍了一种在成年斑马鱼 (斑马斑马) 中产生阿霉素诱发的心肌病模型的方法。本文介绍了两种不同的腹腔注射方法, 并讨论了减少各实验组差异的条件。
Abstract
基因可接触的成年斑马鱼 (斑马斑马) 已越来越多地被用作一种脊椎动物模型, 用于了解诸如心肌病等人类疾病。由于它的方便和顺从的高通量遗传操作, 生成的获得性心肌病模型, 如阿霉素诱发心肌病 (DIC) 模型的成年斑马鱼, 正在打开新的研究途径的大门,包括通过前基因筛选发现心肌病修饰语。不同于早期的斑马鱼 dic 模型, 早期急性和晚期心肌病的慢性阶段可以确定在成年斑马鱼 dic 模型, 使研究阶段依赖的信号机制和治疗策略。但是, 即使在经验丰富的调查人员手中, 也可以通过当前模型获得可变结果。为了促进 dic 模型的未来实施, 我们提出了如何在成年斑马鱼中生成这种 dic 模型的详细协议, 并描述了两种不同的腹腔 (IP) 注射方法。我们进一步讨论如何减少变化, 以获得可靠的结果, 并提出如何正确解释结果的建议。
Introduction
阿霉素 (DOX), 也命名为阿凡霉, 已发展为抗肿瘤药物自二十世纪六十年代1,2。它现在仍然积极地作为一种重要的化疗药物的广泛的肿瘤。然而, DOX 的临床应用因其剂量依赖性毒性而受到阻碍, 特别是心脏症状, 从无症状心电图改变到心包炎和失代偿型心肌病等。1,2. 迄今为止, 至少有三个主要假说被提出来解释 DIC, 包括活性活性氧 (ROS)1、3、4、5、抑制拓扑异构酶 II β (TOP2β)6,7, 和调制细胞内钙释放1,8,9。积累的证据还表明, 遗传易感性是 DIC10,11,12,13的关键危险因素。然而, 与这些 DIC 倾向相关的基因标识仍然很不清楚。右雷佐生是美国食品药品监督管理局 (FDA) 批准的唯一的佐剂, 用于治疗 DIC, 但实施14、15、16的限制, 强调需要确定其他治疗策略。因此, 为这些目的探索了 DIC 的动物模型。由于它们的易接近性和简单, 对 DIC 模型的机械研究可能会对其他类型的心肌产生更广泛的影响: 常见的发病机制可能在不同病因的心肌中共享, 特别是在以后病理阶段17,18,19,20。
除了 DIC 的啮齿动物模型外, 还开发了具有较高吞吐量的斑马鱼 DIC 模型, 以促进发现新的遗传因素和治疗方法。在透明斑马鱼胚胎中建立了一个胚胎 DIC 模型, 用于筛选治疗化合物21。鉴于心肌是成人发病疾病的渐进性发病机制, 成年斑马鱼心肌病模型已发展22,23,24,25,26。我们产生了第一个获得的模型, 由慢性贫血引起的心肌病24, 其次是 DIC 作为第二获得性心肌病模型在成年斑马鱼23。我们发现, 注射一丸的 DOX 到成年斑马鱼诱导心脏, 包括一个急性期大致在1周后注射 (), 其次是慢性心肌病期长达6月后注射。haploinsufficiency 雷帕霉素(mtor) 变好心肌病在慢性期的机械靶向性, 在急性期夸大鱼类死亡率, 强调成人 DIC 模型对分期依赖性的鉴别价值机制23。我们进一步证明, 成人 DIC 模型可以用来强调收集的斑马鱼插入心脏 (ZIC) 突变体正在通过座子的插入突变方法产生27。一个先导屏幕确定了3已知的心肌病基因以及 DnaJ (Hsp40) 同源、亚乙型和成员 6b (dnajb6b) 作为新的 DIC 易感性基因28。因此, 在斑马鱼的成人 DIC 模型的产生导致了一个新的方法, 系统地使鉴定的 DIC 基因修饰语, 这补充了现有的全基因组的关联研究 (GWAS) 和数量性状的轨迹 (QTL分析.
在成年斑马鱼 DIC 模型的生成和实施过程中, 我们注意到不同研究者和/或甚至在同一调查者的不同注射中有显著的差异。模型的纵向性质对注册不同调查人员的结果和顺序的故障排除过程提出了挑战。为了便于研究界使用这种简单的心肌病诱发应力方法, 我们详细描述了我们的协议, 提出了两种类型的 IP 注入, 并讨论了减少不同研究者之间差异的考虑因素。
Protocol
此处描述的所有程序都是按照《实验室动物护理和使用指南》 (国家科学院出版社 2011) 进行的, 并获得梅奥诊所机构动物护理和使用委员会的批准。 1. 成年斑马鱼的准备 在交叉槽中设置足够的繁殖对, 至少要获得 DOX 注射液所需的总鱼的两倍。如果比较不同遗传背景的鱼类, 在同一周内养殖所有鱼类, 以确保与年龄相符的控制。 第二天早上收集鱼胚, 将它们转移?…
Representative Results
本文给出了在成年斑马鱼中对 DIC 模型进行 IP 注射的两种方法。在使用经典的, 建立的 IP 注入方法29, 它指出, 注入 DOX 溶液 (红色) 有时可能会渗出的地方, 针穿透。替代的 IP 注入使用不同的位置, 针穿透, 是3-4 毫米远离腹膜的 DOX 被释放 (图 1A), 这有效地防止泄漏 (图 1B, 1C)。成功地交付 DOX 入腹膜?…
Discussion
为了模拟渐进性 DIC, 20 毫克/千克 DOX 的剂量以实验的方式确定为最高剂量, 在1的过程中不会导致大量的鱼死亡, 但仍然导致鱼死亡和4度后心脏功能的减少 (图 3和图 4C)。这种剂量可与啮齿动物 DIC 模型中常用的 (15-25 毫克/千克) 和极限累计剂量 (550 毫克/米2, 相当于15毫克/千克)<sup class="…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到部分支持, 由美国心脏协会 (14SDG18160021) 的科学家发展补助金, 美国 NIH R01 赠款 hl 81753 和 hl 107304 到 xx, 和梅奥基金会 xx。
Materials
| Crossing tank | Aquaneering | ZHCT100 | Fish breeding |
| Incubator | ThermoFisher | Maintaining embryo | |
| 3 L medium tank | Aquaneering | ZT280 | Maintaining fish |
| Paramecia | Carolina | 131560 | Food for juvenile fish |
| Live hatched brine shrimp | in house | Food for adult fish | |
| Doxorubicin hydrochloride | Sigma | D1515-10MG | |
| 1.5 ml safe-lock tube | Eppendorf | No. 022363204 | For drug storage |
| Aluminum foil paper | Fisher | 1213104 | For preventing light exposure |
| Proteinase K | Roche | No. 03115887001 | For dechorionating embryo |
| Hank's balanced salt solution (HBBS) | ThermoFisher | 14025076 | Vehicle for DOX |
| 100 mm petri dish | Falcon | 431741 | |
| 10 μL NanoFil micro-syringe | WPI | NANOFIL | For injection |
| 34 gauge needle | WPI | NF34BV-2 | For injection |
| Tricaine | Argent | MS-222 | Anesthetizing fish |
| 96 well plate | Costar | 3539 | For embryo drug treatment |
| Transfer pipette | Bel-art product | F37898 | For transfering embryo |
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Cite This Article
Ma, X., Ding, Y., Wang, Y., Xu, X. A Doxorubicin-induced Cardiomyopathy Model in Adult Zebrafish. J. Vis. Exp. (136), e57567, doi:10.3791/57567 (2018).