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人类结肠癌细胞DNA损伤与修复福西的免疫荧光成像
Chapters
Summary June 9th, 2020
Please note that all translations are automatically generated.
Click here for the English version.
在中子捕获治疗（BNCT）中使用的中子混合束激活的辐射诱导DNA损伤反应尚未完全确定。该协议提供了一个分步程序，通过使用中子混合束照射后，通过人体结肠癌细胞系中的免疫荧光染色来检测修复蛋白质的辐射诱导 foci （RIF）。
Transcript
该协议提供了一个分步程序，通过与中子混合束照射后，通过人体结肠癌细胞系中的免疫荧光染色来检测修复蛋白质的辐射诱导源。免疫荧光成像是一种敏感方法，它为修复途径蛋白在 foci 中的外观提供视觉证据，以响应 DNA 损伤剂，如电离辐射。中子混合束用于中子捕获治疗BNCT，辐射引起的DNA损伤反应尚未完全确定。
我们的协议也可用于分析细胞水平的生物效应，由于其他高 LET 束的辐射，如质子使用质子束治疗或使用强子疗法的碳离子。辐照后，从附着的细胞中取出介质，用2.5毫升PBS洗涤一次。然后用一毫升70%乙醇固定细胞10分钟。
要渗透细胞，去除乙醇，用2.5毫升PBS清洗。在PBS中轻轻加入1毫升2%Triton X-100，覆盖盖玻片，并在室温下孵育细胞5分钟。用 PBS 洗涤细胞三次，用 PBS 中稀释的 2%BSA 的一毫升来阻断渗透。
然后用2%BSA孵育细胞至少30分钟。要染色细胞，请按照文本手稿中的建议，加入 PBS 中稀释的正抗体的适当量。然后盖上培养皿，并放在一个塑料盒与保湿木质素，以保持湿度。
在37摄氏度下孵育30分钟。孵育后，用PBS进行三次洗涤，并加入适当量的二级抗体。将盘子与保湿木质素一起回到塑料盒中，并在37摄氏度下孵育至少30分钟。
然后用PBS重复洗涤，并添加100微升DAPI稀释到每毫升1微克的最终浓度，以对抗核。在室温下孵育细胞最多两分钟，然后用PBS重复洗涤。最后一次洗涤后，取出 PBS，轻轻将盖玻片放在安装介质的顶部，避免形成气泡。
用指甲油密封盖玻片的边缘，在进行荧光显微镜之前，使安装介质变硬。Gamma-H2AX foci是DNA双链断裂的标准标记物，在中子混合、光束辐照和非辐照结肠癌细胞中被检测。foci 显示为明显的荧光点。
在辐照细胞中观察到伽马-H2AX foci的直径较高。此外，还检测到高-LET阿尔法粒子的单个轨迹穿过核。免疫荧光显微镜用于检测代表性修复蛋白Rad52和DNA依赖蛋白激酶。
与对照细胞相比，在照射后DNA断裂时测量了DNA依赖性蛋白激酶叶径的高均值。在辐照细胞中观察到聚类DNA双链断裂，其强度更高，为复杂、较大和聚类。免疫荧光染色最重要的阶段是细胞固定和细胞膜渗透。
这些步骤是需要让抗体很容易渗透到这些细胞和细胞内修复蛋白质。此过程提供有关在细胞级别激活每个修复路径的信息。然后，使用实时PCR等测定方法，在分子水平上确认结果。
这项技术使研究人员有可能探索辐射引起的DNA损伤反应，而对于使用抗癌疗法的不同光束，这种反应还没有完全确定。
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