单细胞凝胶电泳技术检测小鼠成纤维细胞DNA的氧化损伤及修复

Oxidant-induced DNA damage and the reparation in NIH3T3 mouse fibroblasts by single cell gel electrophoresis

目的观察小鼠成纤维细胞由过氧化氢(H2O2)引起的DNA损伤及其修复,并详细介绍单细胞凝胶电泳技术。方法培养NIH3T3细胞,H2O2造成细胞氧化损伤,单细胞凝胶电泳技术(SCGE,Comet Assay)检测细胞DNA的损伤情况。结果①建立了H2O2致NIH3T3细胞DNA损伤的分级图谱;②H2O2引起的小鼠成纤维细胞DNA单链断裂与H2O2的浓度呈依赖性关系;③细胞在除去H2O2后15 min已出现明显修复,多数修复可在1 h内完成,但少数修复可能需要较长时间才能完成。结论单细胞凝胶电泳技术是一种简便、敏感的检测DNA氧化损伤的方法。

Objective To study H2O2-induced single strand breaks（SSB） formation in DNA and the reparation in NIH3T3 mouse fibroblasts by single cell gel electrophoresis technique （SCGE, Comet Assay） and to introduce SCGE. Methods The NIH3T3 mouse fibroblasts were incubated, and cell damage was induced by H2O2 and determined by SCGE. Results ① H2O2-induced damage class graph of the DNA Comet in the NIH3T3 mouse fibroblasts was established. ② H2O2-induced SSB was H2O2 dose-dependent. ③ The reparation of the damaged DNA was time-dependent and could be achieved 15 min after incubation. The data indicated that most reparations were completed within 1 hour, but a few might take longer time. Conclusion Single cell gel electrophoresis technique is a simple and sensitive method to determine oxidation-induced single strand breaks（SSB） formation in DNA.