在小鼠模型中将胶质母细胞瘤治疗干细胞的鼻内递送
Summary
干细胞是有希望的治疗载体，以治疗脑肿瘤由于其内在的肿瘤向性。非侵入性鼻内干细胞传播绕过血脑屏障，并且具有很强的临床翻译潜力。本文概述了胶质瘤小鼠模型中鼻内干细胞传递的基本原理。
Abstract
对脑恶性肿瘤的内在向性使得干细胞成为治疗恶性肿瘤的有希望的载体。通过鼻内途径递送治疗性干细胞是最近发现的替代策略，由于与颅内植入或通过系统途径递送相比，其具有非侵入性，具有很强的临床翻译潜力。缺乏血脑屏障进一步加强了进行鼻内入侵的干细胞的治疗潜力。本文概述了我们研究中使用的基本技术，并概述了使用颅内神经胶质瘤异种移植小鼠模型进行干细胞传播的鼻内策略的基本原理。我们展示优化的程序，通过具体的预定实验参数产生一致和可重现的结果，并为精简的工作流程提供准则，确保有效执行和可靠的实验总结。该文章旨在作为基于假设，干细胞类型或肿瘤特异性的进一步实验定制的基准。
Introduction
人类干细胞的低毒性，低免疫原性和内在的脑肿瘤趋向性是用于递送治疗剂的有吸引力的特征1 。用于恶性脑肿瘤的新型基于干细胞的治疗剂是近年来发展的有希望的创新，并且该治疗策略的鼻内适应代表了临床翻译的飞跃，因为非侵入性和反复给药可能大大降低患者应用的障碍，可适用于门诊服务，无需全身麻醉或与侵入性外科手术1,2,3,4相关的长期住院服务。
我们和其他人开创了干细胞传播到脑肿瘤的鼻内途径，并为一些基本原则奠定了基础使用小鼠异种移植模型2,3,4的翻译研究，以及通过磁共振成像（MRI）试剂载体研究干细胞在体内的迁移。通过这些试点探索，我们积累了丰富的经验，并获得了如何使用建立良好的恶性胶质瘤患者来源的异种移植（PDX）小鼠模型建立强大的临床前评估策略的洞察力，同时保持调查决议，以检查精细的机械细节，传递到鼻腔的治疗性干细胞的鼻内入脑的复杂生物现象。在这里，我们描述标准化操作方案的原理，以证明使用完善的人神经干细胞系HB1.F3.CD 5的实验研究的现状<sup>， 6,7,8 ，其可以容易地修改以适应使用人干细胞作为治疗载体的特定肿瘤模型或策略。
Protocol
所有动物手术必须经机构动物护理和使用委员会（IACUC）批准或等同。如果本文所述的具体步骤有任何不确定性，请勿继续。向机构的IACUC和指定的兽医工作人员澄清。 1.确保培养细胞的无菌性，并遵循无菌技术原理在给予小鼠9,10之前，遵循所有细胞培养程序和IACUC细胞测试和无病原体状态确认的标准良好实验室实践。 遵循NIH要求进行细胞鉴?…
Representative Results
低氧预处理（ 图4A ） 4和CXCR4过表达（ 图4B和4C ） 4显着上调了CXCR4受体的细胞膜存在，如流式细胞术所证实的。 NSCs表现出的肿瘤趋向性（蓝色箭头）显示在肿瘤组织组织学（红色圆圈）中。通过普鲁士蓝染色确认肿瘤中MPIO标记（ 图4D ）干细胞的存在（ <strong class="x…
Discussion
虽然药物递送的鼻内途径已被广泛地用于小分子，纳米医学和蛋白质化合物18 ，但是用于鼻内脑肿瘤靶向的治疗性干细胞的应用在开发中的脑肿瘤治疗剂谱2 中是非常新的， 4 。鼻腔干细胞的行为有内在的复杂性，分子细节尚不清楚。干细胞的大小及其与颅神经的分布机制与非细胞生物制剂或小分子显着不同，目前我们正在进?…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到NIH R01NS087990（MSL，IVB）的支持。
Materials
| Stereotaxic frame | Kopf Instruments | Model 900 | |
| Hypoxic Cell Culture Incubator | ThermoFisher Scientific | VIOS 160i | |
| Cell culture supplies (Plastics) | ThermoFisher Scientific | Varies | Replaceable with any source |
| Legend Micro 21R Refrigerated Microcentrifuge | ThermoFisher Scientific | 75002490 | Replaceable with any source |
| Bench centrifuge Sorvall ST16R | ThermoFisher Scientific | 75004240 | Replaceable with any source |
| Micro syringe 702N 25µl (22S/2"/2) | Hamilton Company | 80400 | Flat tip |
| Sample Tray for Irradiator | Best Theratronics | A13826 | To set up mice protection with lead shield |
| Leica DMi8 Microscope | Leica Microsystem | Custom setup | |
| Leica CM1860 UV cryostat | Leica Microsystem | Custom setup | |
| Exel International Insulin Syringe | ThermoFisher Scientific | 14-841-31 | |
| Corning Phosphate Buffer Saline | Corning Cellgro/ThermoFisher | 21-031-CV | |
| Dulbecco's Modified Eagle Medium | Corning Cellgro/ThermoFisher | 11965-084 | |
| Trypsin 0.05% | Corning Cellgro/ThermoFisher | 25300054 | |
| Hyaluronidase from bovine testes | MilliporeSigma | H3506 |
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Citar este artigo
Yu, D., Li, G., Lesniak, M. S., Balyasnikova, I. V. Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model. J. Vis. Exp. (124), e55845, doi:10.3791/55845 (2017).