肺癌细胞系中癌症干细胞的放射敏感性
Summary
癌症干细胞的存在与放射治疗后复发或不良结果有关。本手稿描述了研究肺癌细胞系中癌症干细胞的放射敏感性的方法。
Abstract
癌症干细胞（CSCs）的存在与放射治疗后复发或不良结果有关。研究耐辐射CSC可以为克服无线电电阻提供线索。电压封闭钙通道[2]1亚单位等分形式5被报告为非小细胞肺癌（NSCLC）细胞系中耐辐射CSC的标记物。以钙通道+2+1亚单位为例，提出了NSCLC细胞系中CSC对辐射敏感性的研究方法。CSC按流动细胞测定用假定标记排序，通过球形形成测定评估被分类细胞的自更新能力。殖民地形成测定，确定有多少细胞失去产生后代形成菌落的能力后，一定剂量的辐射，然后进行评估已分的细胞的辐射敏感性。本手稿提供了研究CSC的辐射敏感性的初步步骤，为进一步了解基本机制奠定了基础。
Introduction
放射治疗在癌症治疗中起着重要的作用。然而，放射性抗癌干细胞（CSCs）的存在可能导致放射治疗1、2后复发或不良结果。CSC的特点是其自我更新能力和产生异源性癌细胞的能力3。具有更有效的DNA损伤修复能力或更高水平的自由基清除系统或其他机制的装甲，CSC相对耐放射治疗4，5，6，7,8.识别CSC标记并探索其机制将有助于开发能够克服放射性阻力而不增加正常组织损伤的药物。
电压封闭钙通道[2]1亚单位等分形式5已报告为NSCLC细胞系9中耐辐射CSC的标记。#2_1 最初被确定为肝细胞癌 （HCC）10的 CSC 标记。使用从同一患者的原发性肿瘤和复发性肿瘤中提取的一对HCC细胞系进行减法免疫，鉴定出一种名为1B50-1的抗体，专门针对复发性HCC细胞。1B50-1阳性细胞在体外表现出较高的球形形成效率和体内的高肿瘤原性。其抗原通过质谱鉴定，为钙通道[2]1亚单位等分形式5。_2_1 在CSC中具体表达，在大多数正常组织中无法检测到，使其成为针对CSC10的潜在候选者。#2_1 也可以作为 NSCLC 细胞系的 CSC 标记，并且通过提高 DNA 损伤修复效率来部分向 NSCLC 细胞提供放射性电阻，以响应辐射9。
研究耐辐射CSC可以为克服无线电电阻提供线索。以NSCLC中的β2+1为例，提出了研究CSC辐射敏感性的主要方法。通常，CSC用假定表面标记分离，并比较阳性和阴性细胞群的干细胞特征和放射敏感性。无血清培养基的球形形成，辅以支持自我更新的生长因子，是评估体外细胞干细胞的有益测定。高球形形成能力的细胞在注射到免疫缺陷小鼠10、11、12中时，很可能表现出高肿瘤原性。然后，利用殖民地形成测定来评估细胞的辐射敏感性，从而确定有多少细胞在辐射13后失去了产生形成菌落的后代的能力。
Protocol
Representative Results
Discussion
该协议描述了研究体外癌细胞系中CSCs的放射性敏感性的方法。在本研究中，在NSCLC细胞系中，β2+1的表达是连续的。因此，门控基于等型控件。在分拣之前，应通过流式细胞测定在多个细胞系中检查β2+1表达，并通过QPCR或西方斑点验证。建议通过流式细胞测定、在荧光显微镜下观察荧光或QPCR（排序后），重新分析已排序的β2+1和β2+1低细胞的表达。
球形形成测定是评价自我更?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了国家自然科学基金（81402535和81672969）和国家重点研究开发项目（2016YFC0904703）的支持。
Materials
| 0.5% Trypsin-EDTA (10X), no phenol red | Thermo Fisher | 15400054 | Dilute in to 0.05% (1X) with autoclaved distilled water |
| 1B50-1 | This antibody is produced and friendly supplied by Laboratory of Carcinogenesis and Translational Reseach (Ministry of Education/Beijing), Department of Cell Biology, Peking University Cancer Hospital and Institute. See reference 10. Alternatively, commercial antibody of calcium channel α2δ1 subunit can be used (ABCAM, ab2864) (Yu, et al., Am J Cancer Res, 2016; 6(9): 2088-2097) | ||
| 4% formaldehyde solution | Solarbio | G2160 | |
| A549 | ATCC | RRID: CVCL_0023 | |
| B27 | Thermo Fisher | 17504044 | |
| Biological Safety Cabinet | Thermo Fisher | 1336 | |
| Centrifuge | Eppendorf | 5910R | |
| DMEM/F-12 | Thermo Fisher | 12500062 | |
| EGF Recombinant Human Protein | Thermo Fisher | PHG0311 | |
| Fetal bovine serum | Thermo Fisher | 16140071 | |
| FGF-Basic (AA 1-155) Recombinant Human Protein | Thermo Fisher | PHG0261 | |
| Flow cytometer/cell sorter | BD | FACSARIA III | |
| H1299 | ATCC | RRID: CVCL_0060 | |
| H1975 | ATCC | RRID: CVCL_1511 | |
| Lightning-Link Fluorescein Kit | Innova Biosciences | 310-0010 | |
| linear accelerator | VARIAN | CLINAC 600C/D | |
| Methyl cellulose | Sigma Aldrich | M7027 | |
| Penicillin-Streptomycin, Liquid | Thermo Fisher | 15140122 | |
| Phosphate buffered saline | Solarbio | P1020 | |
| RPMI-1640 | Thermo Fisher | 11875093 | |
| SYBRGREEN | TOYOBO | QPK-201 | |
| TRIzol | Thermo Fisher | 15596026 | |
| Violet crystal staining solution | Solarbio | G1062 |
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