单细胞凝胶电泳联合原子力显微镜观测UVB诱导的细胞DNA损伤模式变化

Visualization of Cellular DNA Damage Mode Induced UVB by Atomic Force Microscope and Comet Assay

目的:检测UVB诱导的真核细胞DNA损伤。方法:采用单细胞凝胶电泳与原子力显微镜。结果:不同照射剂量的UVB引起的真核细胞DNA损伤模式不同。在0～20 J/m2照射剂量范围内DNA无损伤;在20～360 J/m2照射剂量范围内DNA损伤程度加快;当照射剂量超过360 J/m2时DNA损伤速度减慢,实验组之间无显著性差异,出现"平台"。原子力显微镜的观察结果表明随着UVB照射剂量的增加,DNA结构的变化经历了断裂、交联与断裂并存的损伤增强趋势。当照射能量达到280 J/m2时细胞DNA大都形成断片,并相互交联在一起。这一结果表明彗星电泳检测到的UVB照射剂量达到一定剂量后,DNA损伤出现"平台"的原因可能是此时DNA发生了链内或链间交联。结论:不同照射剂量的UVB造成的细胞DNA损伤模式不同;原子力显微镜是一种比较直观的观测DNA损伤的方法。借助原子力显微镜我们可以深入了解单细胞凝胶电泳检测的原理,为DNA损伤检测提供更优良的检测手段。

Objective： The damage of cellular DNA induced by UVB were examined. Methods： Atomic force microscope （AFM） and were applied. Results： The results showed that the mode of cellular DNA damage induced by different doses of UVB were different. Within 0 - 20J/m2 dose no DNA damage; within 20 - 360 J/m2 dose occurs the DNA damage, injury level is proportional to the radiation dose; when the radiation doses greater than 360 J/m2 the DNA damage slowed down, the injuries were no significant differences between groups （P〉 0.05）. Low-energy UVB irradiation, the cellular DNA damage was manifested mainly DNA strand breaks and strand breaks were not serious, accompanied by chain thicker; while high-dose UVB irradiation （280 J/mS）, a large number of cells, DNA fragments, and fragments cross-linked with each other. The results of AFM showed that the reason of the plateau of comet assay might be DNA frag- ments cross-linked with each other. Conclusions： Experimental results show that the cellular DNA damage and damage type induced by UVB were a dose-dependent. AFM is an efficient method to differentiate the types of DNA damage.