人外周淋巴细胞中γH2AX和53BP1的双重免疫荧光
Summary
该协议提出了一种通过同时检测博来霉素处理的人外周淋巴细胞的间期核中的γH 2AX和53BP1病灶来评估DNA双链断裂的形成和修复的方法。
Abstract
双链断裂（DSB）是细胞核中可能发生的最严重的病变之一，如果不修复，它们可能导致严重的后果，包括癌症。因此，该细胞具有修复DSB的复杂机制，并且这些途径涉及组蛋白H2AX在Ser-139（即γH2AX）和p53结合蛋白1（53BP1）处磷酸化形式。由于这两种蛋白质都可以在DSB的位点形成病灶，因此鉴定这些标志物被认为是研究DSB及其修复动力学的合适方法。根据导致γH2AX和53BP1病灶形成的分子过程，研究它们在DSB附近的共定位可能更有用，以便建立一种替代方法，允许通过同时检测两个DNA损伤标记来量化DSB。因此，该协议旨在评估放射性模拟剂博来霉素通过在双重免疫荧光中存在γH 2AX和53BP1病灶诱导的人淋巴细胞中的基因组损伤。使用这种方法，我们还描绘了γH2AX和53BP1病灶数量随时间的变化，作为研究博来霉素诱导的DSB的修复动力学的初步尝试。
Introduction
DNA损伤由内源性物质持续诱导，例如细胞氧化代谢产生的ROS，或外源性，包括化学物质和物理1。在最有害的病变中，双链断裂（DSBs）在导致基因组不稳定方面起着重要作用，因为它们会导致染色体畸变，进而引发致癌过程。因此，细胞被赋予了复杂而有效的DSB修复机制2。
当DSB发生时，细胞触发DNA损伤反应（DDR），其中与MRE11 / RAD50 / NBS1复合物一起招募ATM或ATR激酶以激活减缓或停止细胞周期的其他蛋白质3。这些激酶的一个重要靶标是组蛋白H2AX，它在DSB的几个兆碱基（即γH2AX）内的Ser-139上磷酸化，从而允许募集几种修复因子，例如BRCA1和p53结合蛋白1（53BP1）3。之后，触发同源重组（HR），非同源末端连接（NHEJ）或单链退火（SSA）之间的一种途径来修复DSB4，5。因此，53BP1参与决定HR或NHEJ之间的选择，主要促进NHEJ的激活而不是HR6。此外，H2AX组蛋白和53BP1的磷酸化形式都可以在DSB的位点形成病灶。由于这些病灶持续到双链的完整性恢复，因此在时间间隔内评估γH2AX或53BP1病灶的出现/消失被认为是评估细胞系统中DSB发生和修复的有用方法6，7。然而，根据上述分子过程，由于γH2AX和53BP1病灶预计在DDR8，9期间在DSB附近共定位，因此在双重免疫荧光中同时检测这些标志物的存在是有用的。
因此，本手稿的目的是评估同时定量γH2AX和53BP1病灶的适用性，以评估放射性模拟剂博来霉素诱导的人外周淋巴细胞的基因组损伤。使用相同的方法，我们还尝试根据先前设置的实验程序10描绘博来霉素诱导的DSB的修复动力学。
Protocol
该研究得到了比萨大学伦理委员会的批准，并获得了每个捐赠者的知情和签署同意。 1. γH2AX和53BP1病灶的形成 样品制备和诱变处理通过静脉穿刺从含有肝素锂作为抗凝剂的采血管（例如真空管）中收集健康成年人的全血样本。 为了保证适当的血液样本保存，请在采样后24小时内开始该程序。 将 300 μL 样品加入含有 4.7 mL 完全?…
Representative Results
通过荧光显微镜分析外周淋巴细胞获得的数据使我们能够评估三个主要方面：博来霉素治疗在增加γH2AX和53BP1病灶（以及DSB）数量方面的有效性，由于其诱变作用，两个病灶在DSB位点共定位的程度，以及γH2AX和53BP1病灶的时间过程，以描绘博来霉素诱导的DSB的修复动力学。正如预期的那样，在未处理和处理的细胞之间观察到γH2AX和53BP1病灶的频率非常高，从而证实了博来霉素诱导外周淋巴细胞中DSB?…
Discussion
γH2AX和53BP1病灶的免疫荧光分析是评估细胞系统间期核中基因组损伤的合适方法。该程序有几个关键点可以影响实验的结果，主要是用于固定和透化的试剂，抗体的类型及其稀释因子以及诱变剂的浓度。
维持蛋白质完整性至关重要，因为免疫荧光方法期望识别主要是蛋白质的抗原。在该协议中，甲醇用于固定淋巴细胞。此步骤特别重要，因为酒精通过置换水并导致可溶性蛋白…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢全血献血者和所有采集血液样本的卫生人员。
Materials
| AlexaFluor 568 goat anti-mouse IgG (γ1) | Invitrogen | A21124 | 53BP1 secondary antibody |
| Bleoprim | Sanofi | bleomycin sulfate (mutagen) | |
| Penicillin-streptomycin solution 100X | Euroclone | ECB3001D | antibiotics for culture medium |
| PBS 10X | Termofisher | 14200075 | Phosphate-buffered saline |
| FBS | Euroclone | EC20180L | Fetal Bovine Serum for immunofluorescence |
| Goat anti-rabbit IgG (H+L) DyLight 488 Coniugated | Termofisher | #35552 | γH2AX secondary antibody |
| Mouse anti-53BP1 monoclonal antibody | Merck | MAB 3802 | 53BP1 primary antibody |
| Labophot 2 | Nikon | Fluorescence microscope | |
| P-histone H2AX (Ser139) rabbit antibody | Cell Signaling | #2577 | γH2AX primary antibody |
| Phytohemoagglutinin | Termofisher | R30852801 | component of culture medium |
| Prolong gold antifade reagent with DAPI | Cell Signaling | #8961 | Antifade solution with DAPI for counterstaining |
| RPMI 1640 | Euroclone | ECB9006L | Culture medium |
| Triton-X100 | Sigma | T9284 | Nonionic detergent for permeabilization |
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Citazione di questo articolo
Falaschi, A., Chiaramonte, A., Testi, S., Scarpato, R. Dual Immunofluorescence of γH2AX and 53BP1 in Human Peripheral Lymphocytes. J. Vis. Exp. (197), e65472, doi:10.3791/65472 (2023).