在斑马鱼胚胎氧化应激分析
Summary
Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.
Abstract
高含量的活性氧物种（ROS）可能导致细胞的氧化还原状态对氧化应激条件的变化。这种情况会导致分子（脂质，DNA，蛋白质）的氧化，并导致细胞死亡。氧化应激也影响一些病理状况如糖尿病，视网膜病，神经退行性疾病和癌症的进展。因此，重要的是定义工具不仅在单细胞水平，而且在整个生物体的情况下，调查氧化应激的条件。在这里，我们考虑的斑马鱼胚胎在体内系统中一个有用的执行这样的研究，并提出了一个协议来测量体内氧化应激。利用荧光探针活性氧和斑马鱼转基因荧光线，我们开发两种不同的方法来测量氧化应激在体内 ：I）的“全胚胎ROS的检测方法”对氧化应激的定性测量和ii）“单细胞ROS的检测方法“对氧化应激的定量测量。在此，我们证明这些程序的有效性由氧化剂剂和生理或遗传方式增加氧化应激的组织。该协议是服从正向遗传筛选，这将有助于ROS的地址因果关系中的氧化应激相关的疾病，如神经系统疾病和癌症的动物模型。
Introduction
氧化应激被专门定义为一个条件，结果从不平衡细胞的氧化还原状态。这通常发生在细胞内复杂的氧化还原反应确定细胞的氧化还原状态。氧化还原反应包括的所有化学反应，包括在电子生物分子产生减少和分子（ 即氧化还原反应）的氧化原子之间的转移。这些反应是通过电子方式激活产物（ 即亲氧化性物质），其特征在于，一个极端的结构不稳定性和其与邻近的生物分子交换不平衡的电子的自发活化催化。这些不规则的反应结果放入DNA损伤，蛋白羧化和脂质过氧化，最终导致细胞死亡1。氧化应激水平的增加已经与衰老和不同的病理状态2的进展相关。氧化应激有据报道，负责血管改变在糖尿病和心血管疾病3,4。它还在神经元变性阿尔茨海默病的关键作用和帕金森氏病5。另外，氧化应激已被证明是在管癌的进展和转移的事件6,7的一个关键因素。此外，炎症和免疫反应可能引发进一步的支持氧化应激8。
在活细胞中，亲氧物种是来自氧（ROS，活性氧物种）或氮（RNS;活性氮）。活性氧包括羟基自由基，超氧阴离子（OH）（O 2 – ）和过氧化氢 （H 2 O 2）。主要RNS是一氧化二氮（NO。）可以通过自发的相互作用来产生betwee一系列继发反应物种的ÑROS和RNS或游离金属离子9。例如，超氧阴离子自由基与氮氧化物发生反应，形成peroxynitrate（ONOO – ），而H 2 O 2反应，以Fe 2 +产生的羟自由基。 ROS和RNS，由于其与几种生物分子发生反应的能力，被认为是生理氧化还原状态10维护一个危险的威胁。以保持氧化还原状态的细胞都配备了一系列解毒抗氧化剂分子和酶。超氧化物歧化酶（SOD），过氧化氢 酶，谷胱甘肽过氧化物酶和Peroxiredoxins基本上构成了抗氧化酶法，阿森纳，从亲氧化物质提供细胞保护包括H 2 O 2，OH和过氧化亚硝酸盐– 11。还抗氧化分子，如维生素C和E，多酚和辅酶Q10（辅酶Q10）是至关重要的淬火ROS和他们的危险去衍生参数12,13。然而，过量生产的ROS和RNS，或者在抗氧化剂系统功能障碍，为转移细胞的氧化还原状态的朝向氧化应激14。
除了他们的贬义，活性氧可以在不同来源的细胞发挥不同的生理作用。正常细胞产生的ROS作为信号分子介导正常的生理活动，如宿主防御和修复创面15-17。反应性物质是响应信号因子，生长因子，和钙的水平18,19的胞内波动通常生产中的细胞通过细胞内的酶，如氮氧化物（NADPH氧化酶）和XO（黄嘌呤氧化酶）。据报道，活性氧可能差异调节的重要核因子如p53或细胞组分，如ATM激酶的DNA损伤反应20的主调节器的活性。类似的ROS强烈调解日影响细胞信号Ê氧化和蛋白质酪氨酸磷酸酶酶（PTPs），其被建立为信号转导21的关键调节剂失活。此外，基于蛋白质组学的方法证明RNS还负责特定的蛋白质修饰和分子信号的改变。 RNS反应与半胱氨酸的巯基修饰成S-nitrothiols（SNO）和触发伴随着病理状态，如炎症和自身免疫性疾病，22,23的分子途径。
由于细胞培养实验中只是部分地重现了众多的演技在体内的因素，它是非常感兴趣的动物模型24,25进行氧化还原的研究。为了实现这一目标，在斑马鱼一直被认为是合适的脊椎动物的动物模型来研究氧化应激动力学26。斑马鱼是给予几个优势，在脊椎动物的开发研究细胞和基因活动的新模式系统elopment和疾病。可以生成和提供每周一次实验需要大型集群胚胎。此外斑马鱼胚胎的非凡的光学清晰度，以及它们的小尺寸，使单细胞成像和动态跟踪在整个生物体27。在过去的十年中，已经产生了相当多的斑马鱼突变体来模拟人类的病理状况，如癌症和遗传疾病28-31。最重要的是，大量的转基因株系已经产生，允许基因和生物操纵32的广泛的机会。例如，转基因的组织特异性的斑马鱼线被经常用于体内研究。这些行表示一个选择的启动子的控制下的荧光蛋白质，提供识别单个细胞在体内的功能，以及它们含有的解剖结构。
几个毒理学研究已经采用t他斑马鱼来评估化学品对氧化还原平衡在体内的作用，提示这种脊椎动物的适用性作为动物模型，药物发现和氧化应激33-35的领域。即使一些荧光探针已经过测试，以监测氧化应激的斑马鱼幼虫36,37，有没有既定的分析检测和测量氧化应激在斑马鱼组织中的水平和活细胞。在这里，我们描述了用于在体内量化氧化应激在活斑马鱼胚胎细胞的过程。成像工具，流式细胞仪分选，荧光探针和促氧化条件都结合在一起，生成一个简单的化验检测和氧化物质的定量分析在斑马鱼胚胎和组织。
Protocol
Representative Results
Discussion
关键步骤
对氧化应激的检测在本文中所描述的斑马鱼胚胎的过程包括两个不同的方法。整个安装ROS检测方法主要是定性测定活性氧检测用，而单 细胞ROS的检测方法允许更具体的定量测量（ 图1）。这两种方法提供了一种快速简便的方法来评估对斑马鱼胚胎的体内活性氧检测。然而，他们都提出了一些关键步骤。
关键步骤相关的方法我<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Support in Massimo Santoro lab come from HFSP, Marie Curie Action, Telethon and AIRC. We thank Dafne Gays and Emiliano Panieri for critical reading of the manuscript.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Hydrogen peroxide solution | SIGMA | 516813 | DO NOT STORE DILUITIONS |
| Hank's Balanced Salt Solution 1X | GIBCO | 14025 | |
| Methyl cellulose | SIGMA | M0387 | |
| Instant Ocean Aquarium Sea Salt Mixture | INSTANT OCEAN | SS15-10 | |
| Tricaine | SIGMA | A5040 | |
| Cgeneric ROS-sensitive probe: CellROX Deep Red Reagent | INVITROGEN | C10422 | |
| Mitochondria specific ROS-sensitive probe: MitoSOX | INVITROGEN | M36008 | dissolve one vial with 13μl of DMSO |
| Hydroethidine | INVITROGEN | D23107 | |
| Rotenone | SIGMA | R8875 | Prepare 5mM stock solution in DMSO. |
| Dimethyl sulfoxide | SIGMA | D2650 | |
| VAS2870; 3-Benzyl-7-(2-benzoxazolyl)thio-1,2,3-triazolo(4,5-d)pyrimidine | EnzoLifeScience | BML-EI395 | dissolve the powder in DMSO; diluite in fish water |
| Propidium Iodide | Molecular probes (Life Technologies) | P3566 | |
| 7-aminoactinomycin D (7-AAD) | Molecular probes (Life Technologies) | A1310 | |
| Nrf2a Morpholino | GeneTools | 5'-CATTTCAATCTCCATCATGTCTCAG-3' | Ref: Timme-LaLaragy et al; 2012 (PMID: 22174413); Kobayashi et al; 2002(PMID:12167159 ) |
| Collagenase P | ROCHE | 11213857001 | Dissolve the powder at 100mg/ml in sterile HBSS. Store aliquots at -20°C |
| Phosphate-Buffered Saline (PBS) | GIBCO | 10010-056 | |
| Fetal Bovine Serum | GIBCO | 10082-147 | |
| Complete Protease Inhibitor Cocktail Tablets | ROCHE | Dissolve one tablet in 1ml of water | |
| 0.5% Trypsin-EDTA (10x), no phenol red | GIBCO | 15400-054 | Prepare 1X working solution before usage |
| Compound microscope | ZEISS | ||
| Stereo microscope with fluorescent illumination | Nikon | AZ100 | |
| camera | ZEISS | AxioCamMRm | |
| software for fluorescence image acquisition | ZEISS | ZEN 2011 | |
| Fluorescence-activated cell sorter | BD FACSCalibur | ||
| Centrifuge | Eppendorf | 5417R | |
| FACS tubes | BD | 342065 | |
| Multiwell Plate | BD Falcon | 353047 | |
| Sterilized, non treated Petri dishes 90mm | VWR | 391-1915 | |
| Confocal microscope | Leica | Leica SP5 |
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