小鼠体内合成蛋白的定量测量
Summary
脊髓液蛋白水平升高可能是血浆蛋白在改变的血脑屏障上扩散的结果，也可以是血脑内合成的结果。本文提出了一种优化的测试方案，有助于区分这两种情况，并提供对内代谢蛋白的定量测量。
Abstract
脑脊液（CSF）是一种存在于大脑和脊髓中的液体，对基础科学和临床科学都非常重要。CSF蛋白质成分的分析为基础神经科学研究以及神经系统疾病提供了关键信息。需要注意的一点是，在CSF中测量的蛋白质可能同时来自血清的术中合成和转位，而CSF的蛋白质分析只能确定这两个组分的总和。为了区分动物模型和人类血液内产生的蛋白质转位和蛋白质，CSF蛋白质分析测量使用传统的蛋白质分析工具必须包括白蛋白CSF/血清商（Q白蛋白）的计算，这是血脑界面（BBI）完整性的标志，以及蛋白质指数（Q蛋白/Q白蛋白），这是钙蛋白内合成的估计。该协议说明了从CSF和血液采集到商数和指数计算的整个过程，用于定量测量神经疾病小鼠模型中的细胞内蛋白质合成和BBI损伤。
Introduction
脑脊液（CSF）是一种围绕大脑和脊髓的透明无色液体，具有重要的临床和基本科学意义。CSF保持中枢神经系统（CNS）的电解环境，平衡全身酸碱状态，为神经元和胶质细胞提供营养，作为中枢神经系统的淋巴系统发挥作用，并在CNS1中传输激素、神经递质、细胞因子和其他神经肽。因此，由于CSF成分反映了中枢神经系统的活性，这种流体提供了一个有价值的，但间接的，以表征的中枢神经系统的生理和病理状态。
CSF已经用于诊断影响中枢神经系统的疾病超过一百年，在大部分时间，它主要是由临床医生作为诊断工具研究。然而，近年来，神经生物学家已经认识到CSF在研究中枢神经系统病理生理学方面的潜力。特别是，神经科学领域引入了几种高通量蛋白分析工具，以便对CSF的蛋白质组成进行详细研究，并期望这种分析有助于深入了解动态变化发生在CNS内。
Luminex和Simoa技术2，3等多倍免疫测定技术的技术发展，使今天的研究人员能够以极低的浓度检测数百种蛋白质。此外，这些相同的技术允许使用小样本量，从而促进对小动物，包括小鼠的研究，在小动物中，CSF的有限样本量直到最近才排除了对液体的详细特征。
然而，一个警告是，在CSF中测量的蛋白质可能来自细胞内合成和/或从血清转出由于血脑界面（BBI）受损。不幸的是，仅CSF的蛋白质分析只能确定这两个成分的总和。为了区分转体蛋白和内蛋白，必须根据血清浓度的个体变异性以及屏障完整性调整使用任何可用蛋白质分析工具的CSF蛋白测量。然而，虽然这种调整在临床实践中是常用的，例如CSF IgG指数，该指数对检测细胞内IgG合成4、5、6具有高灵敏度，但迄今为止很少有研究能够纠正CSF蛋白浓度对血清浓度和屏障完整性的7、8。
目前，Reibergram方法是确定蛋白质屏障功能和宫内合成的最佳方法。它是CSF/血清商图中的图形化评估，以综合的方式分析屏障（dys）功能和宫内蛋白合成，指纯血衍生蛋白质9，10。高度丰富的蛋白质白蛋白通常被选择作为参考蛋白，因为它只在肝脏中产生，因为它的大小，大约70kDa，是小蛋白和大蛋白质11之间的中间体。分析图最初由赖伯和费尔根豪尔于1987年对免疫球蛋白（Igs）11的主要类别进行定义，根据对数千个人类样本9的分析结果进行实证分析。该方法随后被两个菲克扩散定律应用于分子扩散/流速理论12得到证实。这种理论证明蛋白质通过屏障扩散具有双曲分布，可以定量解释中枢神经系统9、13中蛋白质的动态。总体而言，使用 Reibergram 来演示片内蛋白合成的优点是，它同时识别从血清进入 CSF 的蛋白质分数，以及由于本地生产而在 CSF 中发现的蛋白质量。
本文和相关协议描述了从CSF和血液采集到最终计算校正CSF蛋白水平，用于神经小鼠小鼠模型中的细胞内蛋白合成的定量测量。障碍。此过程提供了一个基准，用于评估 （1） 任何 CSF 蛋白质的病理生理学来源和 （2） 屏障完整性的稳定性和功能意义。这个程序和协议不仅可用于评估小鼠CSF样本，而且可用于分析多种动物模型的神经系统疾病和人类患者的CSF。
Protocol
所有动物工作都采用达特茅斯盖塞尔医学院机构动物护理和使用委员会 （IACUC） 审查和批准的协议。 1. 流体收集 注：血清和CSF均是必需的。生存和尸体解剖需要每个流体收集两个协议。 使用生存程序收集血清和CSF注：对于生存液体收集，血清收集应先于CSF收集，因为它是一个侵入性较低的程序。CSF 必须在血清抽取后一周内获得。血清?…
Representative Results
这项具有代表性的实验旨在比较多发性硬化症（MS）两个临床相关啮齿动物模型中IgG的宫内合成：PLP139-151-诱导的复发性自免疫性脑脊髓炎（R-EAE）和慢性渐进性、泰勒的小鼠脑脊髓炎病毒诱发脱骨髓性疾病（TMEV-IDD）。R-EAE是理解复发性MS的有用模型，而TMEV-IDD模型具有慢性渐进性MS19。 在本研究中，对R-EAE（n = 12）和TMEV-IDD（n = 28）中的宫内IgG合成进?…
Discussion
评估CSF蛋白质浓度增加的定量方法是中枢神经系统生理和病理状态表征的有用工具。然而，除了可靠的CSF蛋白质水平定量外，CSF蛋白的检测还需要表达在CSF中区分血液和CNS衍生成分的结果。然而，迄今为止，常用的蛋白质定量测定不允许区分两种蛋白质成分，即使借助高通量工具也是如此。因此，为了区分在CNS隔间中合成的蛋白质和从血液中提取的蛋白质，提出了对蛋白质测量的具体校正。这种?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者感谢达特茅斯的比较医学和研究中心（CCMR）的工作人员对用于这些研究的小鼠进行专家护理。伯恩斯坦研究基金资助了这项研究。
Materials
| 1 mL insulin syringe | BD | 329650 | |
| 1 mL syringe | BD | 329622 | |
| 25 gauge needle | BD | 305122 | |
| 3 mL syringe | BD | 309582 | |
| 30 gauge insulin needle | BD | 305106 | |
| Absorbent pads | Any suitable brand | ||
| Acepromazine | Patterson Vet Supply Inc | ||
| BioPlex Handheld Magnetic Washer | BioRad | 171020100 | Magnet |
| BioPlex MAGPIX Multiplex Reader | BioRad | 171015001 | |
| BioPlex Pro Flat Bottom Plates | BioRad | 171025001 | |
| Biotinilated detection antibody | Any suitable source | The antibody has to be directed against the species of the protein of interest. | |
| Bovine Serum Albumin (BSA) | Sigma | A4503 | |
| Buprenorphine hydrochloride | PAR Pharmaceutical | NDC 42023-179-05 | |
| Capillary Tubes | Sutter Instrument | B100-75-10 | OD: 1.0 mm, ID: 0.75 mm Borosilicate glass 10 cm; drawn over Bunsen to make ID smaller. |
| Centrifuge tube, 0.2 mL | VWR | 20170-012 | |
| Centrifuge tube, 0.5 mL | VWR | 87003-290 | |
| Centrifuge tube, 1.5 mL | VWR | 87003-294 | |
| Chlorhexidine diacetate | Nolvasan | E004272 | |
| Disposable pipettes tips | Any suitable brand | ||
| Ear bars | KOPF Instruments | 1921 or 1922 | |
| Ethanol | Kopter | V1001 | |
| Freezer | VWR | VWR32086A | |
| Gauze | Medline | NON25212 | |
| Heating pad | Sunbeam | XL King Size SoftTouch, 4 Heat Settings with Auto-Off, Teal, 12-Inch x 24-Inch | |
| Induction Chamber | VETEQUIP | ||
| Isoflurane | Patterson Vet Supply Inc | NDC 14043-704-06 | |
| Ketamine (KetaVed) | Patterson Vet Supply Inc | ||
| MagPlex Microspheres (antibody-coupled) | BioRad | Antibody-coupled magnetic bead | |
| Microplate Shaker | Southwest Scientific | SBT1500 | |
| Microretractors | Carfill Quality | ACD-010 | Blunt – 1 mm |
| Microsoft Office (Excel) | Microsoft | ||
| MilliPlex MAP Mouse Immunoglobulin Isotyping Magnetic Bead Panel | EMD Millipore | MGAMMAG-300K | Commercial kit for the quantification through Luminex of a panel of immunoglobulin isotypes and subclasses in mouse fluids. |
| Mouse Albumin capture ELISA kit | Novus Biological | NBP2-60484 | Commercial kit for the quantification through ELISA of albumin in mouse fluids. |
| Multichannel pipette | Eppendorf | 3125000060 | |
| Non-Sterile swabs | MediChoice | WOD1002 | Need to be autoclaved for sterility |
| Oxygen | AIRGAS | OX USPEA | |
| Pasteur Pippettes | Fisher | 13-678-20A | 5 & 3/4" |
| PDS suture with disposable needle, 6-0 Prolen | Patterson Vet | 8695G | P-3 Reverse Cutting, 18" |
| PE-Streptavidin | BD Biosciences | 554061 | |
| Pipetters | Eppendorf | Research seriers | |
| Polyethylene tubing | |||
| Refrigerated Centrifuge | Beckman Coulter | ALLEGRA X-12R | |
| Scale | Uline | H2716 | |
| Scalpel | Feather | EF7281 | |
| Shaver | Harvard Apparatus | 52-5204 | |
| Standard proteins | Any suitable source | The best choice for a reference standard is a purified, known concentration of the protein of interest. | |
| Stereotaxic instrument | KOPF Instruments | Model 900LS | Standard Accessories |
| Sterile 1 x PBS | Corning Cellgro | 21-040-CV | |
| Sterile saline | Baxter | 0338-0048-02 | 0.9 % Sodium Chloride Irrigation USP |
| Surgical Forceps Curved, 7 (2) | Fine Science Tools | 11271-30 | Dumont |
| Surgical Scissors | Fine Science Tools | 14094-11 | Stainless 25x |
| Vaporizer + Flow meter | Moduflex Anhestesia Instruments | ||
| Vortex | Fisher | 02-215-414 | |
| Warming pad | Kent Scientific Corporation | RT-JR-20 | |
| Water Sonicator | Cole Parmer | EW-08895-01 | |
| Xylazine | Patterson Vet Supply Inc |
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Cite This Article
Gilli, F., Welsh, N. C., Linzey, M. R., Royce, D. B., DiSano, K. D., Pachner, A. R. Quantitative Measurement of Intrathecally Synthesized Proteins in Mice. J. Vis. Exp. (153), e60495, doi:10.3791/60495 (2019).