用基因编码荧光传感器评估毒理学氧化应激的成像方法
Summary
这篇手稿描述了使用基因编码的荧光记者在一个应用的活体细胞成像检查异诱导氧化应激。这种实验方法提供了无与伦比的时空分辨率、灵敏度和特异性, 同时避免了用于检测毒理学氧化应激的常规方法的许多缺点。
Abstract
虽然氧化应激是一种常用的毒理学机制, 但传统的研究方法存在许多缺陷, 包括样品的破坏、潜在的伪影的引入, 以及缺乏反应的特异性涉及的物种。因此, 目前需要在毒理学领域的非破坏性的, 敏感的, 和具体的方法, 可以用来观察和量化细胞内氧化还原摄动, 更常见的称为氧化应激。在这里, 我们提出了一种方法, 使用两个基因编码的荧光传感器, roGFP2 和超, 将用于活细胞成像研究, 以观察异诱导的氧化反应。roGFP2 均衡与谷胱甘肽氧化还原电位 (EGSH), 而超高直接检测过氧化氢 (H2O2)。这两种传感器都可以通过转染或转导的方式表达成各种细胞类型, 并且可以针对特定的细胞室。最重要的是, 使用这些传感器的活细胞显微镜提供了高的空间和时间分辨率, 这是不可能使用传统的方法。在 510 nm 监测的荧光强度的变化作为两个基因编码的荧光传感器的读数, 当连续兴奋 404 nm 和 488 nm 光。该属性使两个传感器比率, 消除了普通的显微工件, 并纠正了传感器在细胞之间的表达差异。这种方法可以应用于各种荧光平台, 能够激发和收集规定波长的排放, 使其适合于共焦成像系统、常规的广域显微镜和平板读者.两种基因编码的荧光传感器都被用于各种细胞类型和毒理学研究, 以实时监测细胞 EGSH和 H2O2生成。这里概述了一个标准化的方法, 广泛适用于细胞类型和荧光平台的应用 roGFP2 和超高活性细胞毒理学评估的氧化应激。
Introduction
“氧化应激” 这一术语经常被称为毒理学中的一种机制, 但这一术语很少被具体描述。氧化应激可以指几种细胞内的过程, 包括活性氧的生成、自由基的损伤、抗氧化分子的氧化, 甚至是特定信号的激活。范围广泛的环境污染物1,2和药物代理3,4已被记录为通过异化合物本身的直接作用来诱导氧化应激5 ,6或次要地通过生产氧化剂种类作为蜂窝响应的一部分7、8、9、10。因此, 对毒理学有极大的兴趣, 能够准确地观察和描述导致不良结局的氧化过程。测量氧化应激的常规方法包括氧化生物分子的鉴定11,12,13,1415或抗氧化剂16 ,17,18,19,20, 或直接测量活性物种本身21,22,23, 24。但是, 这些方法通常需要蜂窝中断, 这通常会消耗样本, 消除空间分辨率, 并可能引入工件25。开发更灵敏和具体的方法来检测氧化剂种类和氧化应激标志, 广泛适用于研究异暴露的不利影响。
使用新一代基因编码的荧光传感器的活细胞显微镜已经成为监测细胞内氧化还原状态的有力工具。这些传感器通常是使用在病毒启动器的控制下通过转染或转导方法引入的载体来表达的。高表达效率是不必要的, 因为细胞表达的荧光传感器可以很容易地识别视觉。对于毒理学评估, 可以使用荧光显微镜观察表达这些传感器的细胞, 因为它们是实时接触异化合物的。这种实验设计允许在同一个单元中重复测量, 允许每个细胞的既定基线作为自己的控制。活细胞成像所提供的高时间分辨率非常适合于检测氧化事件, 特别是那些规模不大或瞬态性的。除了对它们的目标分子敏感和特异外, 这些传感器的荧光也可以用两种波长的光来激发。这种现象允许将荧光发射表达为一个比值, 它允许识别与真实传感器反应相关的信号变化, 如传感器表达的变化, 细胞厚度, 灯荧光探测器的强度、漂白和灵敏度26。使用荧光传感器的另一个优点是, 它们可以针对特定的蜂窝单元, 创建一个由常规方法25,26,27所无法比拟的空间分辨率级别。
一个以绿色荧光蛋白 (GFP) 为基础的基因编码的大家族被开发出来, 其特征是报告各种生理指标, 包括 pH 值、温度、钙浓度和 ATP/ADP 比值25 ,28,29,30,31。其中包括谷胱甘肽氧化还原电位 (EGSH) 和过氧化氢 (H2O2) 的传感器。虽然这些传感器是为应用于氧化还原生物学和生理学, 他们也已适应研究异诱导氧化应激。具体地说, 此处概述的协议描述了使用 EGSH传感器 roGFP2 和 H2O2传感器超级。
roGFP2 报告的氧化还原电位的细胞内还原和氧化谷胱甘肽 (GSH/GSSG) 通过一个氧化还原继电器涉及谷胱甘肽过氧化物酶 (GPx), 氧 (Grx), 谷胱甘肽还原酶 (GR) (图 1)25,32,33. 谷胱甘肽是主要的细胞抗氧化分子, 并存在于其还原形式 (GSH) 在 millimolar 浓度在胞25,34。虽然 EGSH尚未链接到任何功能结果, 但它被认为是细胞内氧化状态的重要指标34。GSSG 浓度的相对较小的增加会导致 EGSH的增加, 这是由 roGFP2 检测到的。同样重要的是, 在异暴露过程中使用 roGFP2 监测 EGSH , 可能会在氧化还原继电器和相关通路 (如戊磷酸盐分流器) 的几个点上揭示作用机理, 例如,图1)35。这里讨论的第二个传感器, 超, 是一个胞内 h2O2探针, 从插入黄色荧光蛋白 (YFP) 到细菌 H2O2敏感转录的规管领域中获得。因子 OxyR136。虽然它以前被认为是破坏性的活性氧中间体, H2O2越来越被认为是生理条件下的一个重要的细胞内信号分子37,38, 建议在毒理学中也存在用于 H2O2的无法识别的角色。例如, 由异曝光引起的过量的 H2O2可能是蜂窝信号中失调的前兆或生物的转移。
两个基因编码的荧光传感器都已被表达在几个既定的细胞线, 包括人表皮样癌细胞线 A431 和人支气管上皮细胞线 BEAS-2B, 以观察变化的 EGSH和 H2O2以响应各种毒理学暴露。这些包括气态污染物 (臭氧35)、微粒物质的可溶性组分 (12 醌39、40和锌41) 和镍纳米颗粒 (未发布的数据)。这些研究只代表了这两种传感器可能应用的一个小子集。理论上, 任何能够通过常规分子生物学技术接收和表达这些传感器的 DNA 的细胞类型都可以用来评估物质怀疑改变细胞氧化状态的影响。到目前为止, 这些传感器中的一个或多个已经表达了各种原和真核生物, 包括几个哺乳动物, 植物, 细菌和酵母细胞类型25,26,36,42。EGSH和 H2O2传感器的读数是在激发时在 510 nm 发出的荧光强度随488和 404 nm 光的变化。该方法广泛适用于荧光平台, 包括各种类型的显微镜 (共焦和广域) 和平板阅读器。这里提出的方法允许对细胞内 EGSH和 H2O2在体外毒理学系统的敏感和具体观察。
Protocol
Representative Results
Discussion
使用roGFP2 和超检测细胞内 EGSH和 H2O2的变化, 分别在以前的生理和毒理学研究中得到了很好的描述25,35,36 ,39,40,41,42,43。这里概述的协议报告了一种有效的方法来实现这些基?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
作者想感谢凯特 Lavrich 在实验设计和稿件编辑方面的帮助。
Materials
| roGFP2 Plasmid | University of Oregon | N/A | Generous gift of S.J. Remington |
| HyPer Plasmid | Evrogen | FP941 | |
| Hydrogen Peroxide | Sigma Aldrich | H1009 | |
| Dithiothreitol | Sigma Aldrich | 10708984001 | |
| 9,10-Phenanthrenequinone | Sigma Aldrich | 156507 | |
| Black Wall Glass Bottomed Dishes | Ted Pella | 14029-20 | |
| BEAS-2B cell line | American Type Culture Collection | CRL-9609 | |
| Keratinocyte Basal Medium | Lonza | 192151 | |
| Keratinocyte Growth Medium BulletKit | Lonza | 192060 | |
| Excel | Microsoft Office Suite | N/A | |
| NIS-Elements AR Imaging Software | Nikon | N/A | |
| Nikon C1si Confocal Imaging System | Nikon | N/A |
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