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Cancer Research

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

Published: August 25, 2022
doi:
10.3791/64233
, , , , ,
1Tumor microenvironment and animal models lab,Institute of Life Sciences, 2Vascular Biology lab,Institute of Life Sciences, 3Regional Centre for Biotechnology, 4School of Biotechnology,KIIT University

Summary

斑马鱼最近被用作验证潜在辐射改性剂的模型。本方案描述了使用斑马鱼胚胎进行基于辐射的筛选实验的详细步骤以及一些观察方法来评估不同治疗和辐射的效果。

Abstract

斑马鱼被广泛用于多种研究，因为它们是易于维护的脊椎动物模型之一，并表现出独特而方便的模型系统的几个特征。由于高度增殖的细胞更容易受到辐射诱导的DNA损伤，斑马鱼胚胎是辐射研究的一线 体内 模型。此外，该模型在短时间内预测了辐射和不同药物的影响，以及主要的生物学事件和相关反应。一些癌症研究使用了斑马鱼，该协议基于在放疗和癌症背景下使用辐射调节剂。该方法可以很容易地用于验证不同药物对辐照和对照（非辐照）胚胎的影响，从而将药物识别为放射增敏或保护性药物。尽管这种方法用于大多数药物筛选实验，但实验的细节和X射线辐射暴露背景的毒性评估是有限的或仅简要解决，因此难以执行。该协议解决了这个问题，并通过详细说明讨论了程序和毒性评估。该程序描述了一种使用斑马鱼胚胎进行辐射研究和基于辐射的药物筛选的简单方法，具有很高的可靠性和可重复性。

Introduction

斑马鱼（Danio rerio）是一种著名的动物模型，在过去30年中被广泛用于研究。它是一种小型淡水鱼，易于在实验室条件下饲养和繁殖。斑马鱼已被广泛用于各种发育和毒理学研究1,2,3,4,5,6,7,8。斑马鱼繁殖力高，胚胎世代短;这些胚胎适合跟踪不同的发育阶段，在视觉上是透明的，并且适用于各种遗传操作和高通量筛选平台9,10,11,12,13,14。此外，斑马鱼提供实时成像，其发育过程和存在各种有毒物质或因素的不同畸形可以很容易地使用立体或荧光显微镜进行研究7,15,16。

放射治疗是用于治疗癌症的主要治疗方式之一 17,18,19,20,21,22,23,24。然而，癌症放射治疗需要潜在的放射保护剂来保护正常健康细胞免于死亡，同时杀死恶性细胞或在涉及高能辐射的治疗期间保护人类健康 25,26,27,28,29。相反，有效的放射增敏剂也正在研究中，以提高辐射杀死恶性细胞的效率，特别是在靶向和精准治疗中30,31,32,33。因此，为了验证有效的放射防护剂和增敏剂，高度征求了一种适用于半高通量药物筛选并可测量地表现出辐射效应的模型。几种可用的模型用于辐射研究并参与药物筛选实验。然而，高等脊椎动物，甚至是最常用的体内模型小鼠，都不适合大规模的药物筛选，因为用这些模型设计这种筛选实验既费时又费钱，而且具有挑战性。同样，细胞培养模型是各种高通量药物筛选实验的理想选择34,35。然而，涉及细胞培养的实验并不总是实用的、高度可重复的或可靠的，因为培养中的细胞可能会根据生长条件和动力学显着改变其行为。此外，各种细胞类型显示出不同的辐射敏化。值得注意的是，2D 和 3D 细胞培养系统并不代表整个生物体的情况，因此，获得的结果可能无法概括实际的放射毒性水平36,37。在这方面，斑马鱼在筛选新型放射增敏剂和放射防护剂方面具有多种优势。斑马鱼易于操作、离合器尺寸大、寿命短、胚胎发育快、胚胎透明、体型小等特点，是大规模药物筛选的合适模型。由于上述优点，可以在短时间内轻松重复实验，并且可以在多孔板的解剖显微镜下轻松观察效果。因此，斑马鱼在涉及辐射研究的药物筛选研究中越来越受欢迎38,39。

斑马鱼作为筛选辐射调节剂的真正模型的潜力已在各种研究中得到证明 40,41,42,43,44,45。潜在的放射性修饰剂，如纳米颗粒DF1、氨磷汀（WR-2721）、DNA修复蛋白KU80和ATM，以及移植的造血干细胞，以及放射增敏剂，如黄酮吡醇和AG1478，在斑马鱼模型中的作用已被报道19,41,42,43,44,45,46.使用相同的系统，在全身和器官特异性水平上评估了DF-1（富勒烯纳米颗粒）的辐射防护作用，并进一步探索了使用斑马鱼胚胎进行放射防护剂筛选47。最近，据报道，Kelulut蜂蜜是斑马鱼胚胎中的放射保护剂，并被发现可以提高胚胎存活率并防止器官特异性损伤、细胞DNA损伤和细胞凋亡48。

同样，在高通量筛选中检查了通过Hantzsch反应产生的聚合物对斑马鱼胚胎的辐射防护作用，并且主要通过保护细胞免受DNA损伤来提供保护49。在之前的一项研究中，使用这种方法的斑马鱼模型发现亲脂性他汀类药物氟伐他汀是一种潜在的放射增敏剂50。同样，金纳米颗粒被认为是一种理想的放射增敏剂，并已用于许多研究51,52。

斑马鱼的胚胎发育涉及最初 3 小时内的卵裂，其中单细胞受精卵分裂形成 2 个细胞、4 个细胞、8 个细胞、16 个细胞、32 个细胞和 64 个细胞，这些细胞很容易用立体显微镜识别。然后，它达到具有 128 个细胞（受精后 2.25 小时，hpf）的胚泡阶段，其中细胞每 15 分钟翻一番，并经历以下阶段：256 个细胞 （2.5 hpf）、512 个细胞 （2.75 hpf），并在短短 3 小时内达到 1,000+ 个细胞（图 1）。在4小时时，卵达到球形阶段，随后在胚胎质量7,53,54中形成圆顶形。斑马鱼的原肠形成从 5.25 hpf54 开始，在那里它达到盾牌阶段。盾牌清楚地表明细胞向胚环一侧的快速收敛运动（图1），并且是胃胚的一个突出而独特的阶段，可以很容易地识别53,54。尽管胚胎的辐射暴露可以在其发育的任何阶段进行，但原肠胚形成期间的辐射暴露可能具有更明显的形态变化，有助于更好地读出辐射诱导的毒性55;同样，最早可以在2 HPF54开始对胚胎施用药物。

Protocol

本研究是在布巴内斯瓦尔生命科学研究所机构动物伦理委员会事先批准并遵循其指导方针的情况下进行的。所有斑马鱼的维护和繁殖均在28.5°C的常温养鱼设施中进行，胚胎在28.5°C的生物需氧量（BOD）培养箱中维持。 在这里，使用斑马鱼AB品系，并根据Kimmel等人54进行分期。在 6 hpf（屏蔽阶段）给予 X 射线辐射，并在 120 hpf 之前观察到不同的表型。 1. 育?…

Representative Results

该协议的整体布局如图2所示。通过以下分析评估了辐射的影响和剂量依赖性表征。 X射线诱导毒性的评估使用体视显微镜，在药物治疗和/或放疗后评估和表征以下异常。根据经合组织指南61，对于鱼类的毒性评估，包括四个主要的顶端终点，包括胚胎凝固、体节形成畸形、尾巴不脱离卵黄囊以及心跳减少或消失，以分析总体?…

Discussion

斑马鱼在许多研究中被用作有价值的模型，包括几种类型的癌症研究。该模型为大规模药物筛选提供了有用的平台67,68。与任何其他毒性评估方法一样，辐射和/或药物治疗后主要生物学变化的定量评估是该协议最关键的部分。在这类研究中，生存率不能是观察毒性的唯一标准;它需要通过适当的评分系统来评估身体或发育缺陷。在这种情况下，在高达72 h…

Disclosures

The authors have nothing to disclose.

Acknowledgements

SS 的实验室和 RKS 的实验室由印度 DBT 和 SERB 资助。APM是印度政府ICMR奖学金的获得者。DP是印度政府CSIR奖学金的获得者。联合国是印度政府DST-Inspire奖学金的获得者。 图 2 是使用 Biorender （https://biorender.com） 生成的。

Materials

6 Well plates Corning CLS3335 Polystyrene
B.O.D Incubator Oswald JRIC-10
Calcium Chloride Fisher Scientific 10101-41-4
Dissecting Microscope Zeiss Stemi 2000
External Tank for the 1.0 L Breeding Tank Tecniplast ZB10BTE Polycarbonate
Glass petriplates Borosil 3165A75 Glass
GraphpadPrism GraphPad Software, Inc. Version 5.01
Kline concavity slides Himedia GW092-1PK Glass
Magnesium Chloride Sigma-Aldrich M8266
Methylene blue hydrate Sigma-Aldrich 66720-100G
Parafilm Tarsons 380020 Paraffin film
Pasteur pipettes Himedia PW1212-1X500NO Polyethylene plastic
Perforated Internal Tank for the 1.0 L Breeding Tank Tecniplast ZB10BTI Polycarbonate
Polycarbonate Divider for the 1.0 L Breeding Tank Tecniplast ZB10BTD Polycarbonate
Polycarbonate Lid for the 1.0 L Breeding Tank Tecniplast ZB10BTL Polycarbonate
Potassium Chloride Sigma-Aldrich P5655
Sodium Chloride Sigma-Aldrich S7653-5KG
Sodium hydroxide pellet SRL 1949181
Stereo Microscope Leica M205FA Leica Model/PN MDG35/10 450 125
X-Rad 225 Precision X-Ray Precision X-Ray X-RAD 225XL
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Zebrafish LarvaeRadiation ScreeningRadiosensitizersRadioprotectorsRadiation-based ExperimentsHigh-throughput Drug ScreeningRadiobiologyRadiotherapyX-ray IrradiationEmbryo StagingObservational ApproachesSurvivalHatching EfficiencyDevelopmental StagesPhenotypic Changes

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Mohapatra, A. P., Parida, D., Mohapatra, D., Nayak, U., Swain, R. K., Senapati, S. Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors. J. Vis. Exp. (186), e64233, doi:10.3791/64233 (2022).
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