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自体影像学作为一种简单而有力的药理靶标可视化和表征方法

Published: March 12, 2019
doi:
10.3791/58879
, , ,
1Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,University of Copenhagen, 2Neurobiology Research Unit and CIMBI,Copenhagen University Hospital, 3Department of Clinical Physiology, Nuclear Medicine & PET,Copenhagen University Hospital

Summary

自体影像学方法通常用于研究放射线与组织切片的结合, 以确定定性或定量药理学。

Abstract

体外自动摄影的目的是想象实验动物和人类对组织感兴趣的蛋白质的解剖分布。该方法是基于辐射寡核苷酸及其生物目标的特定结合。因此, 冷冻组织切片用辐射和溶液孵育, 然后通过检测放射性衰变来定位与目标的结合, 例如, 使用光敏膜或荧光粉成像板。由此产生的数字自动测底图显示出显著的空间分辨率, 从而能够在不同的解剖结构中对辐射寡核苷酸进行定量和定位。此外, 定量允许通过离解常数 (kd)、抑制常数 (k i) 以及选定组织中的结合位点密度 (b最大值) 对配体亲和力进行药理鉴定.因此, 该方法提供了有关目标定位和配体选择性的信息。在这里, 这项技术的例子是在哺乳动物脑组织中高亲和力γ-羟基丁酸 (ghb) 结合位点的自体成像表征, 特别强调了有关结合试验的方法学考虑。参数、辐射点的选择和检测方法。

Introduction

自动摄影是一种提供放射性衰变图像的方法。该技术通常用于研究一种感兴趣的蛋白质在体外的组织分布, 其基础是放射性标记化合物与其靶点之间的特定药理相互作用。这提供了有关目标配体选择性的直接信息。体外自体影像学也可用于定量测定放射性药物的药理结合参数, 如离解常数 (kd) 和结合位点密度 (b最大值), 以及用于测定竞争配体1,2的抑制常数 (k i)。与传统的同质辐射结合相比, 自动影像学具有能够可视化空间解剖和给出区域表达模式简洁细节的优点 3。因此, 自体影像学方法是免疫细胞化学的相关替代方法, 尤其是在没有经过验证的抗体的情况下。在标准的放射性同位素实验室中, 由于有合适的放射位基因和所需的药理特异性, 可以使用用于制备组织切片的微孔低温恒温器, 以及适当的成像, 因此可以很容易地在标准的放射性同位素实验室进行自体测药能够分析各自组织切片中放射性分布的装置。值得注意的是, 辐射寡核苷酸的一个重要选择标准是与非目标地点的结合数量有限。这可以是其他蛋白质, 膜或材料, 如塑料或过滤器, 并统称为非特异性结合。通常情况下, 非特异性结合是不饱和的, 但如果它涉及特定的非目标蛋白, 则可以是饱和的。验证真正的特定结合的最佳方法是将缺乏目标的组织进行比较,例如基因工程 (淘汰赛) 组织4。

在这里, 该方法说明了与自交图表征的高亲和力结合位点的γ-羟基丁酸 (ghb) 在哺乳动物大脑中。了解 ghb 与其结合部位之间的药理作用具有重要意义, 因为 ghb 既是治疗嗜睡和酒精中毒的临床有用药物, 也是哺乳动物大脑的天然成分和娱乐药物6。高亲和力 ghb 结合位点首次被描述使用 [3h] ghb 结合大鼠大脑均质7。多年来, 进一步的自体影像学研究 [3h] ghb 和类似 [3h] ncs-382 已显示高密度结合位的前脑区域的大鼠 8,9,10, 小鼠9猪11和猴子人的大脑 12。然而, 这些结合位点的分子特性和确切的功能相关性仍然难以捉摸。

为了进一步确定结合位点的特征, 并便利对 ghb 的生理作用进行研究, 开发了含有不同亲和力的不同同位素的多种放射性寡头 ([3h] ghb, [3h] ncs-382、[3h] hopcca 和 [125i] bnoph-ghb)13、14、15、16(17 审查)(图 1)。选择性高亲和力放射性和非常高的组织密度的结合结合结合结合, 使使用荧光粉成像技术9,11的高质量图像得以产生。除了在建立自动摄影实验的实际要点和一个例子来说明细节的情况外, 讨论部分将强调 (一) 放射性核素的选择, (二) 检测条件的选择, 以及 (三) 荧光粉的使用成像板与 x 射线胶片的对比。本文的总体目标是提供有关自体影像学技术的技术、方法和科学细节, 以了解蛋白质靶的组织分布和药理分析。

Protocol

所有动物处理都是按照丹麦动物实验监察局的准则进行的。 注: 此处描述的协议包括组织准备 (即小鼠脑组织)、体外自动影像学检测, 以便在新的实验室中建立该方法、暴露于荧光粉成像板以及随后对自动影像学进行密度分析 (图 2), 目的是在不同的解剖结构中定位和定量放射性寡核苷酸结合。为了进行组织学比较, 包括了环己基紫罗兰色染?…

Representative Results

使用所述协议, 利用小鼠大脑中的放射性 ghb 模拟 [3h] hocpca 对高亲和力 ghb 结合位点的解剖分布进行了可视化, 该模拟被切割成冠状、矢状和水平部分 (图 3).).在海马和皮层中观察到高度的结合, 在纹状体中较低的结合, 在小脑中没有发现结合, 这与以前报告的高亲和力 ghb 位点9、10的表达模式相对…

Discussion

自体影像学检测的质量通常是由放射线的敏感性决定的。一个主要的促成因素是选定的放射性同位素, 它是根据已知配体的供应情况或特定标签技术产生具有适当特定活性 (即放射性的数量) 的配体的可行性得出的每单位摩尔的一个半径)23和有限的化学降解量。大量已知配体的放射性被贴上了9、10、24<sup…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

这项工作得到了 lundbeck 基金会 (grant r133-a12270) 和 novo nordisk 基金会 (grant nnf0c0028664) 的支持。提交人感谢 alešmarek 博士提供的 [3h] 辐射寡核苷酸。

Materials

Absolute ethanol Merck Millipore 107017
Acetic acid Sigma-Aldrich A6283
BAS-TR2040 Imaging Plate GE Healthcare Life Science 28956481 20×40 cm – Sensitive to tritium
Cresyl violet acetate Sigma-Aldrich C5042-10G
DPX (non-aqueous mounting medium for microscopy) Merck Millipore 100579
O.C.T. Compound, 12 x 125 mL Sakura 4583 Tissue-Tek
Paraformaldehyde Sigma-Aldrich 16005-1KG-R
Superfrost Plus slides VWR 631-9483 microscope slides
Tissue-Tek Manual Slide Staining Set Sakura Finetek Denmark ApS 4451
Tritium Standard on Glas American Radiolabeld Chemicals, Inc. ART 0123
Xylene substitute Sigma-Aldrich A5597
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Tags

AutoradiographyProtein BindingPharmacological TargetsCryostat SectioningRadioligand LabelingNon-specific BindingSpatial ResolutionDigital ImagingPharmacological Analysis

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Griem-Krey, N., Klein, A. B., Herth, M., Wellendorph, P. Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets. J. Vis. Exp. (145), e58879, doi:10.3791/58879 (2019).
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