Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells
Time-Lapse Monitoring of DNA Damage Colocalized With Particle Tracks in Single Living Cells
复制标题
DOI：
10.1016/j.ijrobp.2016.04.007
复制
发表时间：
2016-09-01
影响因子：
7
通讯作者：
Sawakuchi, Gabriel O.
中科院分区：
文献类型：
--
作者：
McFadden, Conor H.;Hallacy, Timothy M.;Sawakuchi, Gabriel O.
目的：了解强子治疗（HT）束诱导的DNA损伤和修复对于开发新策略以最大限度地使用HT束治疗癌症患者至关重要。然而，时空研究DNA损伤和修复相关的光束能量HT一直具有挑战性。我们报告了一种技术，使时空测量的辐射引起的损伤在活细胞和共定位这种损伤与带电粒子轨道在广泛的临床相关的光束能量。该技术使用新型荧光核径迹探测器，并在光束线中使用荧光共聚焦激光扫描显微镜，以在损伤后数秒内可视化活细胞亚细胞区室内的粒子径迹穿越。方法和材料：我们设计并制作了一台便携式荧光共聚焦激光扫描显微镜，带有荧光标记活细胞的涂层荧光核径迹探测器（HT1080表达标记到XRCC 1（一种单链断裂修复蛋白）的增强的绿色荧光蛋白），将整个组件置于质子治疗束线中，结果：我们成功地获得了质子径迹和DNA单链断裂灶的共聚焦图像。结论：该技术代表了一种创新的方法，用于分析任何HT束线在与治疗相关的能量和剂量率下的生物学反应。它允许精确确定穿过亚细胞区室的轨道数量并监测其中的细胞损伤，并且具有测量来自治疗光束的每个轨道的线性能量转移的潜力。(C)2016 Elsevier Inc. All rights reserved.
Purpose: Understanding the DNA damage and repair induced by hadron therapy (HT) beams is crucial for developing novel strategies to maximize the use of HT beams to treat cancer patients. However, spatiotemporal studies of DNA damage and repair for beam energies relevant to HT have been challenging. We report a technique that enables spatiotemporal measurement of radiation-induced damage in live cells and colocalization of this damage with charged particle tracks over a broad range of clinically relevant beam energies. The technique uses novel fluorescence nuclear track detectors with fluorescence confocal laser scanning microscopy in the beam line to visualize particle track traversals within the subcellular compartments of live cells within seconds after injury.Methods and Materials: We designed and built a portable fluorescence confocal laser scanning microscope for use in the beam path, coated fluorescence nuclear track detectors with fluorescent-tagged live cells (HT1080 expressing enhanced green fluorescent protein tagged to XRCC1, a single-strand break repair protein), placed the entire assembly into a proton therapy beam line, and irradiated the cells with a fluence of similar to 1 x 10(6) protons/cm(2).Results: We successfully obtained confocal images of proton tracks and foci of DNA single-strand breaks immediately after irradiation.Conclusions: This technique represents an innovative method for analyzing biological responses in any HT beam line at energies and dose rates relevant to therapy. It allows precise determination of the number of tracks traversing a subcellular compartment and monitoring the cellular damage therein, and has the potential to measure the linear energy transfer of each track from therapeutic beams. (C) 2016 Elsevier Inc. All rights reserved.