γH2AX 和53BP1 的免疫荧光显微镜分析 DNA 双链断裂的形成和修复
Summary
这篇手稿提供了一个协议的分析 DNA 双断裂的免疫荧光显微镜的γH2AX 和53BP1。
Abstract
dna 双断裂 (争端) 是严重的 dna 损伤。对争端研究组的形成和修复的分析在包括基因组完整性、遗传毒性、辐射生物学、衰老、癌症和药物开发在内的广泛范围内是相关的。针对争端处理, 组蛋白 H2AX 磷酸化在丝氨酸139在一个区域的几个 megabase 对形成离散核病灶检测的免疫荧光显微镜。此外, 53BP1 (p53 结合蛋白 1) 是另一种重要的争端应变蛋白促进修复的争端, 通过 nonhomologous 端加入, 同时防止同源重组。根据γH2AX 和53BP1 的具体功能, 通过免疫荧光显微镜对γH2AX 和53BP1 进行综合分析, 可以为解决争端的详细分析提供一种合理的方法。这份手稿提供了一个 step-by 步骤的协议, 辅以有条不紊的说明来执行这项技术。具体而言, 细胞周期对γH2AX 病灶形态的影响表现在细胞系 NHDF 的正常成纤维细胞中。此外, γH2AX 病灶作为一个生物标志物的价值被描述在一个健康个体的 x 射线照射的淋巴细胞中。最后, 用γH2AX 和53BP1 免疫荧光显微镜对急性髓系白血病患者的 CD34+ 细胞进行遗传不稳定性研究。
Introduction
dna 不断被内源性破坏 (例如, 复制应力, 活性氧种类, dna 的内在不稳定性) 和外源 (如如, 化学自由基, 辐照) 源 (图 1)1,2 ,3,4。在 dna 损伤中, dna 双断裂是特别严重的病变, 可能导致细胞死亡或癌变。每个单元格和单元格周期5可能会出现大约50争端。在哺乳动物细胞中, 同源重组 (HR) 和 nonhomologous 端接 (NHEJ) 被开发为修复争端解决的主要途径 (图 2)。HR 发生在后期 S/G2 阶段, 并使用完整的姐妹染色单体作为一个模板, 潜在的无差错修复。相比之下, NHEJ 是活跃的整个细胞周期和潜在的诱变作为基础对可以添加或切除前结扎的两端。另外, 在 NHEJ 缺陷6,7的情况下, 可选择的最终连接可作为一种慢诱变备份修复机制。
争端解决 H2AX 在丝氨酸139中诱导了组蛋白的磷酸化, 在每一个不同的 megabase 对周围的一个区域。形成核灶被命名为γH2AX 病灶, 并可通过免疫荧光显微镜检测8。γH2AX 促进了对进一步的争端处理反应蛋白的招募, 并参与染色质重塑, DNA 修复, 和信号转导9。由于每一个γH2AX 的焦点被认为代表一个单一的争端, 通过免疫荧光显微镜来定量的争端分析是可能的, 这已被证明在癌细胞系和患者标本10,11,12,13,14. 53BP1 (p53 结合蛋白 1) 是调解争端修复的另一个关键蛋白质。它参与了对争端应对的蛋白质、检查点信号的招募, 以及争端联会的结束15。此外, 53BP1 在争端修复路径选择中起着关键作用。当 HR 被阻止16时, 它将触发对 NHEJ 的修复。考虑到γH2AX 和53BP1 在争端修复中的真正作用, 用免疫荧光显微镜同时分析γH2AX 和 53BP1, 可为精确分析争端的形成和修复提供一种有用的方法。
这篇手稿提供了一个 step-by 步骤的协议, 执行免疫荧光显微镜的γH2AX 和53BP1 在细胞核。具体而言, 该技术适用于细胞系 NHDF 的正常成纤维细胞、健康个体的 x 射线照射的淋巴细胞和急性髓系白血病患者的 CD34+ 细胞。该方法的细节在所提出的结果的背景下被指出。
Protocol
Representative Results
Discussion
γH2AX 和53BP1 的免疫荧光显微镜是分析广泛的研究领域的一个有效方法。影响实验结果的关键参数是细胞周期的阶段, 细胞的固定和性, 抗体的选择, 荧光显微镜的硬件和软件。
细胞周期对γH2AX 病灶形态的影响表现为细胞系 NHDF 正常成纤维细胞的指数生长。与 S 相中分散的泛核γH2AX 斑点 ( 图 5) 相比, γH2AX 病灶在 G0/G1 和 G2 阶段出现为明显的荧光点…
Disclosures
The authors have nothing to disclose.
Acknowledgements
该项目得到了德国 jos é卡雷拉斯白血病基金会 (DJCLS 14 R/2017) 的支持。
Materials
| RPMI medium | Sigma-Aldrich | R0883 | Medium for cell culture |
| Heparin sodium | ratiopharm | PZN 3029843 | Heparin 5,000 I.U. / mL |
| Sodium chloride solution 0.9% | B. Braun | PZN 1957154 | Component of the anticoagulant stock solution |
| Ficoll-Paque Premium | GE Healthcare | 17-5442-02 | Medium for isolation of mononuclear cells |
| Trypsin solution 10X | Sigma-Aldrich | 59427C | Enzyme for dissociation of fibroblasts in cell culture |
| CD34 MicroBead Kit | Miltenyi Biotec | 130-046-702 | Isolation of CD34+ myeloid progenitor cells |
| Diagnostic microscope slides | Thermo Scientific | ER-203B-CE24 | Microscope slides |
| Megafuge 1.0 R | Heraeus | 75003060 | Tabletop centrifuge |
| Cytospin device | |||
| Lid | Heraeus | 76003422 | Lid for working without micro-tubes |
| Cyto container | Heraeus | 75003416 | Cyto container with 2 conical bores |
| Clip carrier | Heraeus | 75003414 | Carrier for holding a cyto container and a slide |
| Support insert | Heraeus | 75003417 | Support insert for holding a clip carrier |
| Triton X-100 (Octoxinol 9) | Thermo Scientific | 85112 | Detergent for permeabilization of cell membranes |
| Potassium hydroxide solution 1M | Merck Millipore | 109107 | Necessary for preparing the paraformaldehyde solution |
| Paraformaldehyde | Sigma-Aldrich | P6148 | Fixation agent |
| Phosphate buffered saline | Sigma-Aldrich | D8537 | Balanced salt solution |
| Chemiblocker | Merck Millipore | 2170 | Blocking agent |
| Mouse monoclonal anti-γH2AX antibody (JBW301) | Merck Millipore | 05-636 | Primary antibody for detection of γH2AX |
| Polyclonal rabbit anti-53BP1 antibody (NB100-304) | Novus Biologicals | NB100-304 | Primary antibody for detection of 53BP1 |
| Alexa Fluor 488-conjugated goat anti-mouse antibody | Invitrogen | A-11001 | Secondary antibody |
| Alexa Fluor 555-conjugated goat anti-rabbit antibody | Invitrogen | A-21428 | Secondary antibody |
| Vectashield mounting medium | Vector Laboratories | H-1200 | Contains DAPI for staining of DNA |
| Axio Scope.A1 | Zeiss | 490035 | Fluorescence microscope |
| Cool Cube 1 CCD camera | Metasystems | H-0310-010-MS | Camera system for digital recording |
| Isis software | Metasystems | Not applicable | Microscope software |
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