单细胞凝胶电泳技术分析过氧化氢对大鼠外周血淋巴细胞DNA的损伤作用

Analysis of DNA damage in peripheral blood lymphocytes of rats induced by H_2O_2 with single-cell gel electrophoresis

目的 观察不同浓度过氧化氢 (2 5～ 2 0 0 μmol/L)分别作用于正常大鼠外周血淋巴细胞不同时间 (5min和 2 0 min)后细胞DNA的损伤情况。方法 利用快速、灵敏的检测细胞 DNA损伤的单细胞凝胶电泳 (SCG)技术结合激光扫描共聚焦显微镜 ,以“彗星样”淋巴细胞出现率 (计数一定量细胞其中彗星样细胞所占的比例 )、总彗星长度 (“彗星”电泳方向上的最大长度 ,即尾长 )和尾距 (尾部 DNA占总 DNA的百分比与头尾部中心间距的乘积 )来评价淋巴细胞 DNA的损伤程度。结果 过氧化氢能剂量依赖性地导致大鼠外周血淋巴细胞 DNA的明显断裂损伤 ,能使“彗星样”淋巴细胞出现率、总彗星长度和尾距显著增加 ,并随着时间的延长损伤加重。结论 提示活性氧或自由基增多可能是造成体内细胞 DNA断裂损伤的重要原因。

To investigate DNA damage in peripheral blood lymphocytes of rats which were incubated with various doses(25～20 μmol/L) of H 2O 2 for different time duration(5 min or 20 min).Methods DNA damage was measured by single cell gel electrophoresis(SCGE) and laser scanning confocal microscopy. The degree of DNA damage was judged by the percentage of lymphocytes with comet like tail, the tail length and tail moment of the cell.Results DNA damage was demonstrated dose dependently in the peripheral blood lymphocytes of rats when the cells were incubated with the H 2O 2 for certain time. The percentage of lymphocytes with comet like tail, tail length and tail moment were aggravated significantly.Conclusions The results indicate that the increasing of reactive oxygen or free radicals may play an important role in the DNA damage.