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Introduction
Protocol
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Biologia

硝基还原酶/甲硝唑介导的消融和MATLAB平台（RpEGEN）研究斑马鱼视网膜色素上皮的再生

Published: March 02, 2022
doi:
10.3791/63658
, ,
1Department of Ophthalmology, Louis J. Fox Center for Vision Restoration,University of Pittsburgh School of Medicine, 2Earth Research Institute,University of California, Santa Barbara, 3Department of Developmental Biology,University of Pittsburgh School of Medicine

Summary

该协议描述了使用转基因斑马鱼模型对视网膜色素上皮（RPE）进行遗传消融的方法。使用药理学化合物调整方案以纳入信号通路调节被广泛详细。开发了一种基于色素沉着的量化RPE再生的MATLAB平台，并进行了介绍和讨论。

Abstract

视网膜色素上皮（RPE）位于眼睛的后部，执行维持邻近视网膜和血管组织的健康和完整性所必需的功能。目前，哺乳动物RPE的修复能力有限，仅限于小损伤，阻碍了理解 体内 RPE再生过程的进展。在这里，提供了一个详细的方法，以促进利用斑马鱼的 体内 RPE修复的研究，斑马鱼是一种能够进行强大组织再生的脊椎动物模型。该方案描述了转基因硝基还原酶/甲硝唑 （NTR/MTZ） 介导的损伤范式 （rpe65a：nfsB-eGFP），其在 MTZ 治疗 24 小时后可消融 24 小时后消融 RPE 的中央三分之二，随后组织恢复。重点放在斑马鱼幼虫的RPE消融上，并概述了测试药理学化合物对RPE再生的影响的方法。还讨论了RpEGEN的生成和验证，RpEGEN是一种MATLAB脚本，用于基于色素沉着自动量化RPE再生。除了活性RPE修复机制外，该方案还可以扩展到RPE变性和损伤反应的研究，以及RPE损伤对邻近视网膜和血管组织以及其他细胞和分子过程的影响。这种斑马鱼系统在识别驱动RPE再生和RPE疾病相关机制的基因，网络和过程方面具有重大前景，其长期目标是将这些知识应用于哺乳动物系统，并最终实现治疗发展。

Introduction

本文描述的方法详细说明了利用斑马鱼幼虫遗传消融视网膜色素上皮（RPE）的方案。RPE延伸到眼睛的后部，位于神经视网膜的分层层和构成脉络膜的脉管系统层之间。营养支持，光毒性光的吸收和视觉周期蛋白的维持只是RPE执行的一些关键功能，这些功能对于维持这些相邻组织的健康和完整性至关重要1。当病变较小时，对哺乳动物RPE的损害是可以修复的2;然而，较大的损伤或进行性退行性疾病所遭受的损害是不可逆的。在人类中，RPE 退行性疾病（例如，年龄相关性黄斑变性 （AMD） 和 Stargardt 病）导致永久性视力丧失，并且由于可用的治疗选择很少，患者生活质量下降。哺乳动物RPE自我修复能力有限，这在RPE再生过程领域造成了知识空白。鉴于斑马鱼在许多不同组织类型中具有强大的再生能力，该方案旨在建立 体内 脊椎动物系统，以促进对内在再生RPE的研究，并揭示驱动该反应的机制。使用此处概述的消融范式，规范的Wnt信号通路3，mTOR通路4和免疫相关反应5 已被确定为RPE再生的关键介质，可能具有重叠的功能。

在这种遗传消融范式中，斑马鱼（rpe65a：nfsB-eGFP）3表达细菌来源的硝基还原酶（NTR / nfsB）基因6在RPE增强子元件rpe65a7的控制下融合到eGFP。消融是通过在斑马鱼系统水中加入前体药物甲硝唑（MTZ）来实现的。硝基还原酶对MTZ的细胞内活化导致NTR / nfsB表达细胞8，9中的DNA交联和凋亡。该技术已广泛应用于斑马鱼中，以消融视网膜10，11，12，13和其他组织的细胞8。这些元素共同实现了诱导细胞消融方法（NTR / MTZ）8，9和荧光标记物（eGFP）的靶向表达（rpe65a）以进行可视化。

还存在其他有趣的 体内 模型，可用于研究RPE14的再生潜力。这些是广泛的，包括两栖动物视网膜切除术后的RPE到视网膜转分化，其中因视网膜再生而失去的RPE细胞被替换15，16;RPE修复损伤后“超强愈合”MRL/MpJ小鼠17;和自发性RPE和视网膜变性18的大鼠模型中RPE增殖的外源性刺激等。体 外 模型，如成人人RPE干细胞（RPESCs）19 也被开发出来。这些模型都是有价值的工具，用于揭示与RPE再生相关的细胞过程（例如，增殖，分化等）;然而，斑马鱼在消融后具有固有的RPE修复能力方面是独一无二的。

虽然这里的方法是为了专注于理解驱动RPE再生的机制， 但Tg（rpe65a：nfsB-eGFP） 系和这种遗传消融方案可用于研究其他细胞过程，如RPE凋亡，RPE变性以及RPE损伤对邻近视网膜和血管组织的影响。消融方案也可以修改以包括药物操作，这是筛选目标信号通路的便捷初步策略。例如，使用 Wnt 反应抑制剂 -1 （IWR-1）20 阻断规范的 Wnt 途径已被证明会损害 RPE 再生3。这里重复了这一点，以指导用户完成药理学操作实验，并作为概念验证，以验证为根据色素沉着的恢复来量化RPE再生而创建的MATLAB脚本（RpEGEN）。与转基因系和消融方案一样，RpEGEN脚本具有适应性，可用于量化RPE中的其他标记物/细胞过程。

Protocol

此处概述的所有方法均符合匹兹堡大学机构动物护理和使用委员会（IACUC）的要求。 1.斑马鱼胚胎收集前的准备 将胚胎培养箱设置为28.5°C。 制备黑色素生成抑制剂N-苯基硫脲（PTU）21，22的25x储备溶液。该储备溶液根据常用配方22进行缩放，1x等于每体积0.003%重量（%w / v）（例如，…

Representative Results

已知抑制规范的Wnt信号通路会显着损害斑马鱼RPE再生使用方案3中描述的遗传消融范式（rpe65a：nfsB-eGFP）和药理学操作方法（IWR-1）。本文重复该实验，验证了一种基于色素沉着的斑马鱼RPE再生量化自动化方法。下面总结的结果涵盖了方案的所有步骤，从受精当天（0 dpf）到使用RpEGEN量化RPE再生。 在斑马鱼幼体中实施RPE消融方案（通过药物操作）<…

Discussion

该协议描述了对RPE进行遗传消融的方法，并研究了幼体年龄斑马鱼的变性和再生机制。该方案也已在成年斑马鱼3 中成功实施，但表征范围较小，这就是为什么幼虫是这里的重点。方案这一部分的关键方面（步骤1-4）包括：1）在黑色素发生开始之前向胚胎添加1.5倍PTU，2）以2-3 dpf对PTU处理的胚胎进行去壳化，3）仔细筛查eGFP，以及4）MTZ基因消融期间水的变化时间。PTU抑制黑色素?…

Declarações

The authors have nothing to disclose.

Acknowledgements

此处描述的工作得到了美国国立卫生研究院的支持（RO1-EY29410至J.M.G，NIH CORE Grant P30-EY08098至眼科）;UPMC 免疫移植和治疗中心（L.L.L. 和 J.M.G.）;和E. Ronald Salvitti眼科研究主席（致J.M.G.）。此外，还获得了韦根眼科奖学金（L.L.L）、匹兹堡眼耳基金会（Eye & Ear Foundation of Pittsburgh）的额外支持，以及纽约州纽约预防失明研究的无限制资助。作者还要感谢Amanda Platt的技术援助，以及Hugh Hammer博士和水上运动人员提供的出色动物护理支持。

Materials

Lab Material/Equipment
2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) Millipore Sigma D9542
6-well plates Fisher Scientific 07-200-83
Conical Polypropylene Centrifuge Tubes Fisher Scientific 05-539-13 Catalog number is for 50 mL tubes
Diamond tip scribing pen Fisher Scientific 50-254-51 Manufactured by Electron Microscopy Sciences, items similar to this part number are adequate
Dimethyl sulfoxide (DMSO) ≥99.7 % Fisher Scientific BP231 Check instiutional chemical waste disposal requirements
Embryo incubator (large) Fisher Scientific 3720A
Embryo incubator (mini/tabletop) Labnet I5110A
Fluorescence stereo microscope Zeiss Axio Zoom.V16 Or similar, with 488 nm excitation laser/filter
Glass Pasteur pipette Fisher Scientific 13-678-4 Manufactured by Corning, non-sterile
InSolution Wnt Antagonist I, IWR-1-endo Millipore Sigma 5.04462 Manufactured by Calbiochem; 25 mM in DMSO; check instiutional chemical waste disposal requirements
Methylene blue (powder) Fisher Scientific BP117-100 Also available as a premade aqeuous solution
Metronidazole (MTZ) Millipore Sigma M3761 Check instiutional chemical waste disposal requirements
N-phenylthiourea (PTU) Millipore Sigma P7629 Check instiutional chemical waste disposal requirements
Paraformaldehyde (16 % w/v) methanol free Fisher Scientific AA433689M Chemical waste, proper disposal required
Petri dishes Fisher Scientific FB0875712 10 cm diameter
Phosphate buffered saline (powder packets) Millipore Sigma P3813 Used to make 10 X PBS stock
Pronase Millipore Sigma PRON-RO
Shaking incubator Benchmark H2010 Used for incubating MTZ for 1 hour at 37 degrees Celcius
Stereo microscope Leica S9i Or similar, with transmitted light illumination
Student Dumont #5 forceps Fine Science Tools 91150-20 Fine-tipped forceps for manual dechorionation
Tabletop rotator/shaker Scilogex SK-D1807-E
Transfer pipette Millipore Sigma Z135003 3.2 mL bulb draw, non-sterile
Tricaine methanesulfonate (MS-222) Pentair TRS1, TRS2, TRS5 Also available from Fisher Scientific (NC0342409)
VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories H-1200
Software Material
FIJI (Fiji is Just ImageJ) FIJI (Fiji is Just ImageJ) https://imagej.net/software/fiji/ Version: 2.0.0-rc-69/1.52p; Build: 269a0ad53f; Plugin needed: Bio-Formats
GRAMM examples and how-tos MathWorks https://www.mathworks.com/matlabcentral/fileexchange/54465-gramm-complete-data-visualization-toolbox-ggplot2-r-like.
MATLAB MathWorks https://www.mathworks.com/products/get-matlab.html Toolboxes needed to run RpEGEN: Image Processing Toolbox, Curve Fitting Toolbox, Statistics and Machine Learning Toolbox
MATLAB support MathWorks https://www.mathworks.com/support.html
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Referências

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Tags

ZebrafishRetinal Pigment EpitheliumRPERegenerationNitroreductaseMetronidazoleMATLABRpEGENAblationTransgenicEGFPPTUPharmacological Treatment
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Leach, L. L., Fisher, G. B., Gross, J. M. Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium. J. Vis. Exp. (181), e63658, doi:10.3791/63658 (2022).
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