使用线性加速器进行体外放射生物学实验
Summary
临床直线加速器可用于确定各种剂量率对癌细胞的生物效应。我们讨论如何为癌症干细胞样细胞的测定和测定建立线性加速器,这些细胞在悬浮物中生长为肿瘤球体,细胞系作为附体培养体生长。
Abstract
放射治疗仍然是癌症管理的基石之一。对于大多数癌症,它是最有效的,非手术治疗去散体肿瘤。在这里,我们描述了一种用线性加速器照射癌细胞的方法。直线加速器技术的发展提高了放射治疗的精度和效率。广泛的辐射剂量和剂量率的生物影响仍然是一个紧张的研究领域。使用线性加速器可以促进这些研究使用临床相关的剂量和剂量率。
Introduction
放射治疗是治疗多种癌症的有效手段,治疗1、2、3、4。超高剂量照射在放射治疗中是相对较新的,并且由于线性加速器5的最新技术进步而成为可能。超高剂量率比标准剂量率照射的临床优势包括缩短治疗时间和改善患者体验。线性加速器也为基于细胞培养的辐射生物学研究提供了临床环境。辐射剂量和剂量率的生物和治疗影响一直是放射肿瘤学家和生物学家的兴趣,第6,7,8。但是,超高剂量照射和闪光照射的放射生物学-一个极高的辐射剂量率-还有待彻底调查。
伽马射线照射被广泛应用于基于细胞培养的辐射生物学9、10、11。辐射是通过从衰变的放射性同位素源(通常是Cesium-137)发射的伽马射线来实现的。放射源的使用受到高度管制,而且往往受到限制。使用源辐照,很难测试广泛的剂量率,限制了其在分析临床可实现剂量率12的生物效应的效用。
有几个研究说明剂量和剂量率效应12,13,14,15,16,17。在这些研究中,使用了放射性同位素产生的伽马辐照或线性加速器产生的X射线。使用了代表肺癌、宫颈癌、胶质细胞瘤和黑色素瘤的各种细胞系。辐射对细胞生存、细胞周期抑制、凋亡和DNA损伤的影响被评估为读出12,13,14,15,16,17.在这里,我们描述了一种通过使用线性加速器提供基于 X 射线的辐射来定义临床相关辐射剂量和剂量率的生物效应的方法。这些研究应该由生物学家、放射肿瘤学家和医学物理学家密切合作进行。
Protocol
1. 悬浮细胞培养的细胞制备 在细胞培养箱中培养的干细胞培养基培养基培养基细胞约5×106细胞/10厘米板,CO2为5%,37°C为95%相对湿度。注: 细胞培养条件在所有过程中都相同。协议中使用的介质是完整的介质。 在预定辐照前两天,用无菌的5mL移液器从培养板收集胶质瘤干细胞,放入培养罩中的15 mL离心管中。 在台面离心机中,以 200 x g将收集的细胞离心 3 ?…
Representative Results
为了研究标准剂量率和线性加速器照射超高剂量率的细胞周期效应,使用该协议制备了三个胶质瘤干细胞样本,并在辐照17后收集了24小时:一个对照样本未辐照(图2A),一个样品照射400 MU/min(监测单位,4.2 Gy/min标准剂量率,图2B)至4 Gy,另一个样品照射2100 MU/min(21.2 Gy/min超高剂量率,图 2C…
Discussion
放射治疗是癌症管理不可分割的一部分。正在进行的努力旨在提高放射治疗的疗效和效率。直线加速器技术的进步为以前所未有的准确性和安全性治疗患者提供了机会。由于大多数患者都使用直线加速器的高能 X 射线进行治疗,因此,在线性加速器上执行大量剂量率的生物学影响的研究可能很容易应用于患者。已经有一些报告将线性加速器应用于辐射生物学研究,但结果好坏参半,还需要进行13、14、15…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
我们感谢克利夫兰诊所放射肿瘤学系使用线性加速器。我们感谢杰里米·里奇博士慷慨地赠送了胶质瘤干细胞。这项研究得到了克利夫兰诊所的支持。
Materials
| Material | |||
| glioma stem-like cell 4121 | gift from Dr. Jeremy Rich | ||
| 293 cells | ATCC | CRL-1573 | |
| neuron stem cell culture media | Thermo Fisher Scientific | 21103049 | NeurobasalTM media |
| DMEM | Thermo Fisher Scientific | 10569044 | |
| Fetal Bovine Serum | Thermo Fisher Scientific | 16000044 | |
| Penicillin/Streptomycin | Thermo Fisher Scientific | 15140-122 | |
| Recombinant Human EGF Protein | R&D Systems | 236-EG-01M | |
| Recombinant Human FGF basic | R&D Systems | 4114-TC-01M | |
| B-27™ Supplement | Thermo Fisher Scientific | 17504044 | |
| Sodium Pyruvate | Thermo Fisher Scientific | 11360070 | |
| L-Glutamine | Thermo Fisher Scientific | 25030164 | |
| Tripsin-EDTA | Thermo Fisher | 25200056 | |
| extracellular proten matrix | Corning | 354277 | MatrigelTM |
| Ethanol | Fisher chemical | A4094 | |
| Equipment | |||
| 10 cm cell culture dish | Denville | T1110 | |
| 3.5 cm cell culture dish | USA Scientific Inc. | CC7682-3340 | |
| 22x22mm glass cover slip | electron microscopy sciences | 72210-10 | |
| 15 ml centrifuge tube | Thomas Scientific | 1159M36 | |
| 50 ml centrifuge tube | Thomas Scientific | 1158R10 | |
| 5 ml Pipette | Fisher Scientific | 14-955-233 | |
| pipet aid | Fisher Scientific | 13-681-06 | |
| Vortex mixer | Fisher Scientific | 02-215-414 | |
| Centrifuge | Eppendorf | 5810R | |
| Linear Accelerator | Varian | n/a | |
| water equivalent material | Sun Nuclear corporation | 557 | Solid waterTM |
| Reagent preparation | |||
| DMEM media | 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/mL penicillin G, 100 µg/mL streptomycin in 500 ml DMEM media | ||
| stem cell culture media | 10 ml B27 supplement, 20 µg hFGF, 20 µg hEGF, 2 mM L-glutamine, 100 units/mL penicillin G, 100 µg/mL streptomycin in 500 ml Neurobasal media |
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Hao, J., Magnelli, A., Godley, A., Yu, J. S. Use of a Linear Accelerator for Conducting In Vitro Radiobiology Experiments. J. Vis. Exp. (147), e59514, doi:10.3791/59514 (2019).