Ruprecht Nico, Hungerbühler Martin N, Böhm Ingrid B, Heverhagen Johannes T
Department of Diagnostic, Interventional, and Pediatric Radiology, Inselspital, Bern University Hospital, University of Bern, Freiburgstrasse 10, 3010, Bern, Switzerland.
Department of BioMedical Research, University of Bern, Bern, Switzerland.
Radiat Environ Biophys. 2019 May;58(2):295-302. doi: 10.1007/s00411-019-00778-1. Epub 2019 Feb 24.
Currently, in the context of radiology, irradiation-induced and other genotoxic effects are determined by visualizing DSB-induced DNA repair through γ-H2AX immunofluorescence and direct counting of the foci by epifluorescence microscopy. This procedure, however, neglects the 3D nature of the nucleus. The aim of our study was to use confocal microscopy and 3D reconstructed images to improve documentation and analysis of γ-H2AX fluorescence signals after diagnostic examinations. Confluent, non-dividing MRC-5 lung fibroblasts were irradiated in vitro with a Cs-137 source and exposed to radiation doses up to 1000 mGy before fixation and staining with an antibody recognizing the phosphorylated histone variant γ-H2AX. The 3D distribution of γ-H2AX foci was visualized using confocal laser scanning microscopy. 3D reconstruction of the optical slices and γ-H2AX foci counting were performed using Imaris Image Analysis software. In parallel, γ-H2AX foci were counted visually by epifluorescence microscopy. In addition, whole blood was exposed ex vivo to the radiation doses from 200 to 1600 mGy. White blood cells (WBCs) were isolated and stained for γ-H2AX. In fibroblasts, epifluorescence microscopy alone visualized the entirety of fluorescence signals as integral, without correct demarcation of single foci, and at 1000 mGy yielded on average 11.1 foci by manual counting of 2D images in comparison to 36.1 foci with confocal microscopy and 3D reconstruction (p < 0.001). The procedure can also be applied for studies on WBCs. In contrast to epifluorescence microscopy, confocal microscopy and 3D reconstruction enables an improved identification of DSB-induced γ-H2AX foci, allowing for an unbiased, ameliorated quantification.
目前，在放射学领域，辐射诱导的和其他遗传毒性效应是通过γ-H2AX免疫荧光可视化DNA双链断裂（DSB）诱导的DNA修复，并通过落射荧光显微镜直接计数病灶来确定的。然而，该方法忽略了细胞核的三维性质。我们研究的目的是使用共聚焦显微镜和三维重建图像，以改进诊断检查后γ-H2AX荧光信号的记录和分析。将汇合的、不分裂的MRC-5肺成纤维细胞在体外用Cs-137源进行照射，并在固定和用识别磷酸化组蛋白变体γ-H2AX的抗体染色之前，暴露于高达1000 mGy的辐射剂量。使用共聚焦激光扫描显微镜观察γ-H2AX病灶的三维分布。使用Imaris图像分析软件对光学切片进行三维重建并计数γ-H2AX病灶。同时，通过落射荧光显微镜目视计数γ-H2AX病灶。此外，将全血在体外暴露于200至1600 mGy的辐射剂量。分离白细胞（WBC）并对其进行γ-H2AX染色。在成纤维细胞中，仅落射荧光显微镜将全部荧光信号视为一个整体，无法正确划分单个病灶，在1000 mGy时，通过手动计数二维图像平均产生11.1个病灶，而共聚焦显微镜和三维重建为36.1个病灶（p < 0.001）。该方法也可用于白细胞的研究。与落射荧光显微镜不同，共聚焦显微镜和三维重建能够更好地识别DSB诱导的γ-H2AX病灶，从而实现无偏倚的、改进的定量分析。
