前庭神经鞘瘤研究的统一方法框架
1Eaton Peabody Laboratories, Department of Otolaryngology,Massachusetts Eye and Ear, 2Department of Otolaryngology,Harvard Medical School, 3Department of Otolaryngology, Vienna General Hospital,Medical University of Vienna, 4Program in Speech and Hearing Bioscience and Technology,Harvard Medical School
Summary
该方案的目标是概述人类外科手术样品在前庭神经鞘瘤和雪旺氏细胞研究中的多个下游应用的收集和处理。
Abstract
前庭神经鞘瘤是小脑桥角最常见的肿瘤，占所有颅内生长的6-8％。尽管这些肿瘤在高达95％的受影响个体中引起感觉神经性听力损失，但这种听力损失的分子机制仍然难以捉摸。本文概述了我们实验室中建立的步骤，以便于收集和处理各种原始人体组织样品用于前庭神经鞘瘤研究的下游研究应用。具体来说，这项工作描述了从手术样本收集，处理和培养Schwann和神经鞘细胞的统一方法框架。这与现在被认为对当前研究至关重要的并行处理步骤相结合：肿瘤和神经分泌物的收集，RNA的保存和从收集的组织中提取蛋白质，用于制备切片的组织固定，d将原代人细胞暴露于腺相关病毒以用于基因治疗。此外，这项工作突出显示了将这种肿瘤收集在一起的独立手术方法，作为从内耳和外淋巴获得人类感觉上皮的独特机会。提供提高实验质量的提示，并突出显示常见的陷阱。
Introduction
前庭神经鞘瘤（VSs）是小脑桥角最常见的肿瘤，诊断为每10万人1例。虽然非转移性，但这些肿瘤严重影响患者的生命质量1,2,3,4,5,6 。受影响的人通常生活在听力损失，耳鸣和听觉丰满的感觉之中。随着肿瘤生长，症状越来越虚弱，导致平衡问题，面部麻痹和其他颅神经功能受损。由于脑干压迫导致的危及生命的并发症也可能发生。
VS的管理选择本质上限于监视静脉肿瘤和立体定向放射治疗或用于生长肿瘤的手术切除<sup class =“xref”> 8。在研究附属医院手术切除这些肿瘤提供了获取和分析患者手术中收集的新鲜肿瘤组织的机会。 VS的一种特殊的手术方法，即翻转切除术，甚至可以从内耳和外淋巴中获得宝贵的人类感觉上皮细胞。
因为VSs来自外周感觉神经（ 即前庭神经），因此将VS相关观察与从适当的对照神经衍生的神经，如人类大耳神经（GAN）进行比较很重要。健康的GANs在腮腺切除术或颈部解剖中经常被牺牲，可用作健康雪旺氏细胞生理学的健壮模型。
因为没有FDA批准的用于治疗或预防散发性VS的药物，研究人员必须澄清潜在的分子机制确定治疗目标的疾病。已显示在VS发病机制中发挥作用的蛋白质包括merlin，血管内皮生长因子（VEGF），环氧合酶2（COX-2），核因子κB（NF-κB），肿瘤坏死因子α（TNF-α） ，表皮生长因子受体（EGFR）和相关信号分子10,11,12,13,14,15,16,17 。
最近的进展已经扩大和改进了原始人类前庭神经鞘瘤和健康神经组织的收集，处理，培养和下游调查方案18,19 。然而，大多数现有协议被设计为容纳准备这种组织用于单个下游研究应用（ 即单独的细胞培养）。本文提出了一个统一的方法框架，用于同时处理多个下游应用的单个初级人类VS或GAN样品：细胞类型特异性培养，蛋白质提取，RNA保存，肿瘤分泌物收集和组织固定。这项工作还详细介绍了人工脑脊液（CSF）和外翻淋巴结切除术期间的外科手术收集和处理，因为这些密切相关的组织可能作为VS的生物标志物的重要来源。最后，该方案提出了培养中原代人VS细胞病毒转导的步骤，作为该组织用于基因治疗的新应用。
Protocol
在手术前获得了收集所有样品的书面知情同意书，根据世界医学协会道德守则（赫尔辛基宣言）进行了实验。研究方案的所有部分均由马萨诸塞州眼科和马萨诸塞州总医院机构审查委员会批准。 注意：以下1-7节设计为在从手术室接收到初级人类VS或GAN样品后依次执行。处理应始终从第1节开始。然后，通常应首先保留RNA（第2部分），然后制备用于收集分泌物（第3部分）和蛋白…
Representative Results
如第5节所述，培养物中的初级人VS细胞可以被视为许多下游研究应用中疾病相关过程的信息模型（ 图1 ）。在第6节中培养的健康雪旺烷细胞提供了直接和有启发性的比较。如下所述，根据这种统一的方法框架处理的VSs和GAN的广泛数据可以在以前发表的12,13,14,15,27中的多篇文章中找到。 ?…
Discussion
该手稿描述了VS研究的统一方法框架，概述了下游研究应用中人VS和GAN标本的同时处理。随着VS研究进入精密医学的时代，以能够回答许多研究问题的形式准备相同的样本将能够发现特定于个体患者的分子，细胞，遗传和蛋白质组学的见解。
通过免疫细胞化学，使用S100标记阳性的细胞比与DAPI核染色阳性的细胞比例，随时间深入评估人雪旺细胞和VS培养物的纯度。因此，推荐在?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家失聪和其他传播障碍研究所的资助R01DC015824（KMS）和T32DC00038（支持JES和SD），国防部授予W81XWH-14-1-0091（KMS），贝塔雷利基金会（KMS） ，Nancy Sayles Day基金会（KMS），Lauer耳鸣研究中心（KMS）和Barnes Foundation（KMS）。
Materials
| BioCoat Poly-D-Lysine/Laminin 12mm #1 German Glass Coverslip | Corning | 354087 | Or prepare coverslips with Corning Laminin (CB-40232) and Cultrex Poly-L-Lysine (3438-100-01) |
| CELLSTAR 15 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile | Greiner Bio-One | 188161 | |
| CELLSTAR 50 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile | Greiner Bio-One | 227261 | |
| CELLSTAR Cell Culture Dish, 60 mm | Greiner Bio-One | 628160 | |
| Collagenase from Clostridium histolyticum, Sterile-filtered | Sigma-Aldrich | C1639 | |
| Costar 24 Well Clear TC-Treated Multiple Well Plates, Sterile | Corning | 3526 | |
| DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride) | Thermo Fisher Scientific | D1306 | |
| DMEM, high glucose, pyruvate, no glutamine, 500 ml | Thermo Fisher Scientific | 10313-039 | |
| DMEM/F-12, 500 ml | Thermo Fisher Scientific | 11320-033 | |
| Dumont #3 Forceps, Dumoxel | Fine Science Tools | 11231-30 | Autoclave prior to use |
| Dumont #5 Forceps, Standard tip, Inox | Fine Science Tools | 11251-20 | Autoclave prior to use |
| Fetal Bovine Serum, qualified, USDA-approved regions, 500 ml | Thermo Fisher Scientific | 10437-028 | Aliquot in 50 ml tubes and store in -20°C freezer |
| Hyaluronidase from Bovine Testes, Type I-S, Lyophilized Powder | Sigma-Aldrich | H3506 | |
| Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone, 33 mm, sterile | EMD Millipore | SLGP033RS | |
| Paraformaldehyde, Reagent Grade, Crystalline | Sigma-Aldrich | P6148 | Prior to use: Establish Standard Operating Procedures based on protocols available online |
| PBS, pH 7.4, 500 ml | Thermo Fisher Scientific | 10010-023 | Autoclave prior to use |
| Penicillin-Streptomycin (10,000 U/ml), 100 ml | Thermo Fisher Scientific | 15140-122 | |
| PhosSTOP Phosphatase Inhibitor Tablets | Roche | 04906845001 | |
| Pierce Protease Inhibitor Tablets | Thermo Fisher Scientific | 88666 | |
| Pipettes and pipette tips, 5/10/25 ml | Variable | Variable | |
| Plastic Homogenization Pestle for 1.5/2.0ml Microtubes | E&K Scientific | EK-10539 | |
| PrecisionGlide Needles, 27 G x 1 1/2 in | BD | 301629 | |
| RIPA Buffer | Boston BioProducts | BP-115 | |
| RNAlater (RNA stabilization solution) | Thermo Fisher Scientific | AM7021 | |
| Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 ml | Eppendorf | 022363719 | Autoclave prior to use |
| Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 ml | Eppendorf | 022363204 | Autoclave prior to use |
| Saline – 0.9% Sodium Chloride Injection, bacteriostatic, 20 ml | Hospira | 0409-1966-05 | |
| Scalpel Blades – #15 | Fine Science Tools | 10015-00 | |
| Schuknecht Suction Tube 24 gauge | Bausch + Lomb | N1698 42 | Useful for the surgical approach (in addition to common otologic surgical instruments) and e.g. a blue surgical marker |
| Specimen Container, OR sterile, 4OZ | Medline | DYND30331H | |
| Stemi 2000-C Stereo Microscope | Zeiss | 000000-1106-133 | |
| Syringe/Needle Combination, Luer-Lok Tip, 5 ml, 22 G x 1 in. | BD | 309630 | |
| Tuberculin Syringe Only, Slip tip, 1 ml | BD | 309659 | |
| Tuberculin Syringe Only, Slip tip, 3 ml | BD | 309656 | |
| Ultrasonic homogenizer, 4710 Series, CV18 probe | Cole-Parmer | CP25013 |
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Cite This Article
Landegger, L. D., Sagers, J. E., Dilwali, S., Fujita, T., Sahin, M. I., Stankovic, K. M. A Unified Methodological Framework for Vestibular Schwannoma Research. J. Vis. Exp. (124), e55827, doi:10.3791/55827 (2017).