原正畸胶质血异草回顾渗透生长和同源脱氢酶 I 突变

Published: January 14th, 2014

DOI:

10.3791/50865

J. Geraldo Valadez¹, Anuraag Sarangi¹, Christopher J. Lundberg¹, Michael K. Cooper^1,2,3

¹Department of Neurology, Vanderbilt University Medical Center, ²Vanderbilt Ingram Cancer Center, Vanderbilt University Medical Center, ³Neurology Service, Veteran Affairs TVHS

恶性胶质瘤是一组具有明显临床和分子特征的高度渗透性胶质肿瘤的异质性群体。原位矫形异种格拉夫特在前科动物模型中重新概括恶性胶质瘤亚型的组织病理学和分子特征。

恶性胶质瘤是一组具有明显临床和分子特征的高度渗透性胶质肿瘤的异质性群体。原位矫形异种格拉夫特在前科动物模型中重新概括恶性胶质瘤亚型的组织病理学和分子特征。为了模拟世卫组织在移植检测中的I级和IV级恶性胶质瘤，人类肿瘤细胞被异种移植到免疫功能低下小鼠的正交部位——大脑中。与利用培养肿瘤细胞的继发性异种移植物相比，人类胶质瘤细胞与被切除的标本分离，在组织培养中无需事先通过移植即可产生原发性异种移植。本报告中的程序详细说明了肿瘤样本准备、颅内移植到免疫功能低下的小鼠、肿瘤移植监测以及肿瘤收获，供随后进入受体动物或进行分析。肿瘤细胞准备需要2小时，外科手术需要20分钟/动物。

恶性胶质瘤是发生在大脑和脊髓的中枢神经系统的原发性胶质肿瘤。胶质瘤由世界卫生组织（世卫组织）根据组织学上与星形细胞、寡头细胞或子细胞的相似性进行分类，然后根据恶性病理特征进行数值分级（I至IV）。最常见的组织学亚型是天体细胞瘤、寡头性腺胶质瘤和混合寡头肌细胞瘤。包括世卫组织二至四级在内的恶性胶质瘤的特点是侵入性生长和对现有疗法的顽固不化。在美国，每年约有15，750人被诊断为恶性胶质瘤，估计有12，740名患者死于这种疾病。这些统计数字突出了恶性胶质瘤的特别致命性，以及提高疗效的重要需要。

癌症模型对于研究肿瘤生物学和疗法至关重要。人类癌细胞系是体外操纵和体外异种移植研究（继发性异种移植^）1的重要第一步。然而，标准癌细胞培养经历表型和基因型转换^2-4，可能无法恢复在继发性异种移植^5。此外，基因改变，如异辛酸盐脱氢酶（IDH）突变^6，独特的干细胞种群⁷和对关键信号通路⁸的依赖，可能会在癌细胞培养中消失。基因组特征可以在癌症领域培养中更好地保持，但仍不能完全反映原肿瘤的基因型^2，3。直接正交移植否定了体外培养的影响，提供了适当的微环境，并保留了肿瘤启动细胞^9，10的完整性。因此，原生异种移植物是一种强大而相关的临床前模型，用于严格测试靶....

1. 肿瘤细胞悬架的准备

注：需要为使用患者材料和动物提供适当的机构批准，以建立和维持原发性正畸胶质瘤异种移植物。在范德比尔特大学医学中心，在患者同意下收集超过诊断所需量的肿瘤材料，用于研究组织库。标本标本标注带有随机的 5 位 REDcap 数据库编号，并删除所有患者特定标识符。REDcap 数据库包含组织存储库中每个标本的分离临床数据，包括性别.......

分离的胶质瘤细胞被直接移植到免疫功能低下的小鼠的大脑中，以获得原位正畸异种移植线。每个肿瘤标本在移植前被分配一个随机数字，作为去除受保护的健康信息的分离过程的一部分。为此，我们使用 5 位 REDcap 数据库编号。图1 说明了从胶质母细胞瘤（GBM 17182）与同心酸脱氢酶1（IDH1）突变精氨酸132到组织丁（R132H）建立异种移植线的过程和命名。移植后，肿瘤识别号中添加"X"?.......

培养细胞系、异种移植和转基因小鼠是模拟胶质瘤的最常见方法，每个模型系统^3，13，14都有明显的益处和局限性。原位正交胶质瘤异种移植的相关好处包括渗透性生长，这些生长是弥漫性胶质瘤的典型代表，以及保留遗传改变和重要的信号机制，这些机制在培养的胶质瘤细胞中可能极其难以维持。例如，isocitrate脱氢酶突变和声波刺猪配体生产可以维持在原位矫形异种格拉夫特^.......

我们特别感谢范德比尔特大学医学中心的患者，他们为分子神经外科组织库提供了宝贵的研究材料。我们感谢那些建立和维护组织银行的人，里德·汤普森MD（首席调查员），樱桃金纳德RN（研究护士）和拉里A.皮尔斯MS（经理）。组织学服务部分由范德比尔特大学医学中心（VUMC）转化病理共享资源（由 5P30 CA068485 奖支持范德比尔特-英格拉姆癌症中心）执行。这项工作得到了国家发展局（1R21NS070139）、伯劳斯威康基金和VMC发展基金向MKC提供的赠款的支持。MKC 由退伍军人事务部、退伍军人健康管理局、研究和发展办公室、生物医学实验室研究和发展部通过赠款 1 I01 BX000744-01 获得支持。内容不代表退伍军人事务部或美国政府的意见。

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Name	Company	Catalog Number	Comments
Phosphate buffered saline	Life Technologies	14040-133
Papain dissociation system	Worthington Biochemical Corp.	LK003150
Trypan blue solution 0.4%	Life Technologies	15250061
Ketamine HCl	Obtained from institutional pharmacy or local veterinary supply company
Xylazine HCl
Ketoprofen
Ophthalmic ointment
Povidone-iodine	Fisher Scientific	190061617
Cryopreservation medium and proliferation supplement	StemCell Technologies	05751
0.2% Heparin sodium salt in PBS	StemCell Technologies	07980
Penicillin-streptomycin	Life Technologies	15140-122
Dimethyl sulfoxide	Sigma-Aldrich	D6250-5X10ML
NOD.Cg-Prkdc^scid I/2rg^tm1Wjl/SzJ mice	The Jackson Laboratory	005557	NSG mice
Anti-human vimentin antibody	Dako	M7020	Use 1:200 to 1:800
Anti-human IDH1 R132H antibody	Dianova	DIA-H09	Use 1:100 to 1:400

Material	Company	Catalogue Number	Comments
Centrifuge with swinging bucket rotor
Pipetter with dispensing speed control
Disposable hemocytometer	Fisher Scientific	22-600-100
Sterile surgical gloves	Fisher Scientific	11-388128
Disposable gown	Fisher Scientific	18-567
Surgical mask	Fisher Scientific	19-120-1256
Tuberculin syringe	BD	305620
Alcohol pads	Fisher Scientific	22-246-073
Portable electronic scale	Fisher Scientific	01-919-33
Zoom stereomicroscope
Surgical clipper	Stoelting	51465
Scalpel handle	Fine Science Tools	10003-12
Scalpel blades, #10
Stereotaxic instrument	Stoelting	51730
High-speed drill	Stoelting	51449
Drill bit, 0.6 mm	Stoelting	514552
Hamilton syringe	Hamilton	80336
Autoclip, 9 mm	BD	427630
Circulating water warming pad	Kent Scientific	TP-700 TP-1215EA
Hot bead dry sterilizer	Kent Scientific	INS300850
Surgical scissors	Fine Science Tools	14101-14
Fine scissors	Fine Science Tools	14094-11
Spring scissors	Fine Science Tools	15018-10
Dumont forceps	Fine Science Tools	11251-30
Semimicro spatulas	Fisher Scientific	14374
Mouse brain slicer matrix	Zivic Instruments	BSMAS002-1
Cryogenic storage vials	Fisher Scientific	12-567-501

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