立体定向颅内植入和<em在体内</em生物发光成像的肿瘤异种移植的小鼠模型系统的多形性胶质母细胞瘤
Summary
我们描述一个综合性的方法，精确，立体的人多形性胶质母细胞瘤细胞植入到裸鼠的大脑和随后的串行<em在体内</em>成像技术来监测治疗所造成的异种移植物的生长和响应。
Abstract
胶质母细胞瘤（GBM）是一种高品位的原发性脑肿瘤在人类中位生存期仅为14.6个月，尽管标准的三模态的治疗包括手术切除，手术后的放射治疗和替莫唑胺化疗1。显然需要新的治疗方法，以提高患者生存率和生活质量。 GBM的动物模型的，概括人类疾病还允许串行成像技术监测肿瘤的生长和治疗反应，将有助于发展更有效的治疗策略。在本文中，我们描述了我们的技术，精确的立体定向植入生物成像GBM肿瘤细胞的裸鼠移植瘤，概括主要临床特征的GBM 2的大脑。这种方法产生的肿瘤是可重复和精确的解剖位置，同时允许在体内生物发光成像的连续监测内颅移植瘤的生长和治疗3-5。这种方法也很好的耐受性低的围手术期发病率和死亡率的动物。
Protocol
A.术前肿瘤细胞制备混合厚膜U251多形性胶质母细胞瘤细胞稳定表达萤火虫荧光素酶基因的慢病毒表达载体（pGreenFire，系统生物科学）。 这些细胞被生长在10ml完整的Dulbecco的修改的Eagle培养基（DMEM）中，其中包括的DMEM补充10％小牛血清，1％青霉素 – 链霉素，1％非必需氨基酸在一个的T75组织培养烧瓶中孵育5％CO 2和37℃下执行标准细胞培养开始，用洗涤的细胞用磷酸盐?…
Discussion
在本文中所描述的小鼠肿瘤细胞立体定向植入的方法，可重复生成肿瘤的浸润和快速增长的模式，临床多形性胶质母细胞瘤2，6-8，合理地概括。此技术特别适合均匀地不同的可比尺寸和生物学性质，并在特定的解剖位置的重现性肿瘤的治疗组中是可取的分层小鼠的实验。立体定向植入肿瘤细胞的技术，我们描述应该是容易实现的，大多数的翻译研究实验室7,9-11。
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们非常感谢安德鲁·霍兰德博士，萨拉·戴维斯，李舒曼，蒂姆·詹金斯博士祥盛徐的专家协助。我们承认安·肯尼迪博士的支持。 BCB支持的放射生物学培训津贴C5T32CA009677的。 JFD宝来惠康成就奖，医学科学家（1006792）的支持。 JLB支持的超类（5 R25 CA140116-03）资助。我们要感谢史蒂夫哈恩博士的鼓励和支持，使我们的研究可能。我们还要感谢大学的宾夕法尼亚州纳米生物界面中心（NBIC）博士和丹尼斯迪彻的的鼓励和有益的意见。我们认识到，小动物成像设备（SAIF）在美国宾夕法尼亚大学的MRI和光/生物发光核心设施的使用。这些技术开发项目的一部分，由美国国立卫生研究院（RC1 CA145075和K08 NS076548支持01）。
Materials
| Description | Supplier | Catalogue Number | Comments |
| Digital Just for Mouse Stereotaxic Instrument | Stoelting | 51730D | Stereotactic platform for mouse implantation |
| Ketamine/xylazine | Injectable anesthesia | ||
| Puralube Vet Ointment (ophthalmic) | Amazon.com | To prevent drying of the mouse’s eyes | |
| drill holder for the stereotactic platform | Stoelting | 51681 | |
| Micromotor Electric Drill | Stoelting | 51449 | For drilling through the skull |
| .45 mm carbide drill bit | Stoelting | 514551 | |
| Sterile cotton swabs | Fisher Scientific | 23-400-100 | |
| Glass bead dry sterilizer (Germinator 500) | Braintree Scientific | GER-5287 | To sterilize metal surgical instruments |
| Mouse rectal probe | Braintree Scientific | RET-3-ISO | Compatible with the temperature controller |
| Temperature Controller (TCAT-2DF) | Harvard Apparatus | 727561 | Temperature controller to maintain animal’s temperature during surgery |
| Small heating plate | Harvard Apparatus | 727617 | For use with temperature controller to warm mouse during surgery. The heating plate fits under the mouse on the stereotaxic platform. |
| Disposable Scalpels | BD Bard-Parker | 2015-11 | #10 scalpel |
| 10 microliter syringe | Hamilton | 7635-01 | For injection of tumor cells |
| 30 gauge needles, 1″ long, with flat point | Hamilton | Various | Must be compatible with the 10 μl syringe |
| Nanomite Programmable Syringe Pump | Harvard Apparatus | 704507 | Digital motorized syringe injector for stereotaxic device |
| Cellulose sterile surgical spear sponges | Ultracell | 40410 | To dry the surgical field |
| Bone wax | Ethicon | W31 | To seal the burr hole |
| Tissumend II synthetic absorbable tissue adhesive | Veterinary Products Laboratories | 3002931 | To seal the incision |
| Hot water pump with warming pad | Gaymar | TP-650 | Warms mice in post-operative period |
| IVIS Lumina II | Caliper Life Science | Bioluminescent imager | |
| D-Luciferin potassium salt | Gold Biotechnology | LUCK-1 | Luciferin for bioluminescent imaging |
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Tags
Stereotactic Intracranial ImplantationIn Vivo Bioluminescent ImagingTumor XenograftsMouse Model SystemGlioblastoma MultiformeHigh-grade Primary Brain CancerMedian SurvivalStandard Tri-modality TreatmentSurgical ResectionRadiation TherapyTemozolomide ChemotherapyTherapeutic ApproachesAnimal ModelsTumor GrowthTreatment ResponseBio-imageable GBM Cancer CellsNude MiceAnatomic LocationsIn Vivo Bioluminescent ImagingIntracranial Xenograft GrowthPerioperative MorbidityMortality
Citazione di questo articolo
Baumann, B. C., Dorsey, J. F., Benci, J. L., Joh, D. Y., Kao, G. D. Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme. J. Vis. Exp. (67), e4089, doi:10.3791/4089 (2012).