Bulat Tanja, Keta Otilija, Korićanac Lela, Žakula Jelena, Petrović Ivan, Ristić-Fira Aleksandra, Todorović Danijela
Vinča Institute of Nuclear Sciences, University of Belgrade, Belgrade, Serbia.
Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia.
An Acad Bras Cienc. 2016 Mar;88(1):127-36. doi: 10.1590/0001-3765201620140553. Epub 2016 Mar 4.
Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.
电离辐射会诱导DNA双链断裂（DSB），进而引发组蛋白H2AX（γH2AX）的磷酸化。免疫荧光染色可使γH2AX焦点的形成可视化，从而对其进行定量分析。与蛋白质免疫印迹分析和流式细胞术不同，该方法通过显示每个单细胞中荧光信号的确切位置和强度，提供了更准确的分析。实际上，γH2AX的定量分析存在一些问题。本文基于两个问题：对于辐射剂量应采用哪种技术进行测定，以及如何分析由不同显微镜获得的荧光显微镜图像。将HTB140黑色素瘤细胞暴露于剂量范围为1至16 Gy的γ射线下。通过蛋白质免疫印迹分析和免疫荧光显微镜在照射后的不同时间间隔分析对DNA水平的辐射效应。用两种类型的显微镜观察免疫化学染色的细胞：配备ApoTome软件的AxioVision（德国蔡司）显微镜和AxioImagerA1显微镜（德国蔡司）。获得的结果表明，γH2AX的水平与时间和剂量有关。免疫荧光显微镜对于较低辐射剂量能更好地检测DSB，而蛋白质免疫印迹分析对于较高辐射剂量更可靠。配备ApoTome软件的AxioVision显微镜更适合检测γH2AX焦点。
