Baert Philippe, Meesen Geert, De Schynkel Sofie, Poffijn André, Van Oostveldt Patrick
Laboratory for Biochemistry and Molecular Cytology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium.
Micron. 2005;36(4):321-30. doi: 10.1016/j.micron.2005.01.005. Epub 2005 Mar 14.
Analyzing the integrity of DNA is one of the most frequent used endpoints for risk assessment of chemical and physical agents. In the framework of a radiobiological space experiment, this work aimed at having (1) a histochemical tool for the in situ assessment of DNA damage in as long as 20 days old fixed cell cultures, (2) a comprehensive tool for the quantification of different types of DNA lesions, and (3) a methodology of sampling thousands of nuclei based on confocal microscopy, automated stage scanning and digital image processing. For this purpose several fixatives and permeabilization techniques were tested together with the combinatorial use of terminal dUTP transferase-mediated nick end-labeling (TUNEL) and the DNA polymerase I mediated in situ nick translation. These biochemical tools are useful for scoring DNA single and double breaks, and oxidative lesions. Ltk(-) cells were exposed either to hydrogen peroxide or heavy ion beam irradiation. Combination of paraformaldehyde fixation, sodium citrate permeabilization and heat gave the best staining results. A three-channel fluorescence methodology was established including a DNA counter stain for nucleus identification and normalization of DNA content. Communication between confocal imaging software, image analysis software and a relational database proved to be pivotal for a semi-automated high-end single cell analysis and storage of images. In this way, DNA damage data per nucleus can be traced back to the original image. As much as 2500 cells could be analyzed in situ within a day and correlations drawn between different DNA lesion endpoints.
分析DNA完整性是化学和物理因子风险评估中最常用的终点之一。在一项放射生物学空间实验的框架下，本研究旨在：（1）开发一种组织化学工具，用于原位评估长达20天的固定细胞培养物中的DNA损伤；（2）开发一种综合工具，用于定量不同类型的DNA损伤；（3）建立一种基于共聚焦显微镜、自动载物台扫描和数字图像处理对数千个细胞核进行采样的方法。为此，测试了几种固定剂和通透化技术，并将末端脱氧尿苷三磷酸转移酶介导的缺口末端标记（TUNEL）与DNA聚合酶I介导的原位缺口平移联合使用。这些生化工具可用于对DNA单链和双链断裂以及氧化损伤进行评分。将Ltk(-)细胞暴露于过氧化氢或重离子束辐射。多聚甲醛固定、柠檬酸钠通透化和加热的组合产生了最佳染色效果。建立了一种三通道荧光方法，包括用于细胞核识别和DNA含量标准化的DNA复染。共聚焦成像软件、图像分析软件和关系数据库之间的通信被证明对于半自动高端单细胞分析和图像存储至关重要。通过这种方式，每个细胞核的DNA损伤数据可以追溯到原始图像。一天内可原位分析多达2500个细胞，并得出不同DNA损伤终点之间的相关性。
