机械剪切对脾、肝、肾细胞DNA损伤程度的比较

Comparison between effects of mechanical mince on the DNA damages of spleen, liver and kidney cells in mice

目的 探讨机械剪切对小鼠脾、肝、肾 3种细胞DNA的损伤程度是否存在差异以及机械剪切法是否适于 3种器官单细胞的分离。方法 以未经任何处理的小鼠为实验组 ,以 1 5 0mg kg环磷酰胺处理的小鼠为对照组 ,用机械剪切法分离 3种器官的单个细胞并制备单细胞悬液 ,然后用苔盼蓝排斥法检测细胞存活率 ,用单细胞凝胶电泳技术检测细胞DNA损伤程度。结果 机械剪切法能快速分离出彗星实验所需的足量的脾、肝、肾 3种器官的单个细胞 ;试验组脾、肝、肾细胞的拖尾率分别为 3 2 0 %、6 2 1 %和 9 2 2 % ,DNA迁移距离分别为 2 7 30 μm、2 8 4 5 μm和 4 7 1 0 μm ,且不同类细胞之间的拖尾细胞率以及脾细胞与肾细胞、肝细胞与肾细胞之间的DNA迁移距离均有显著差异 (P <0 0 5 )。试验组脾、肝、肾细胞的拖尾率和DNA迁移距离与阳性对照组的同类细胞比较都显著要低 (P <0 0 1 ,P <0 0 0 1 )。结论 机械剪切对小鼠脾、肝、肾 3种细胞DNA的损伤依次加重。机械剪切法对脾。

Objective To study whether there are differences between effects of the DNA damages of the spleen、liver and kidney cells in mice caused by mechanical mince and the spleen、liver and kidney in mice can be dissociated into single cells by mechanical mince or not. Methods No drug was administrated to the tested group mice, and Cyclophosphamide was administrated to the control group mice with 150 mg/kg doses. Then the three kinds of single cell suspensions were prepared by mechanical mince. The cell livabilities were detected by trypanblau coloration, and effects of mechanical mice on the DNA damages of the three kinds of cells were detected by single cell gel electrophoresis. Results The single cell quantity prepared from spleen、liver and kidney in short time by mechanical mince can meet completely the needs of comet assay. The percentages of cells with comets of the tested group were respectively 3 20%,6 21%,9 22% and the DNA migrations were respectively 27 30μm,28 45μm,47 10μm. There were significant differences between the percentages of cells with comets in the tested groups( P <0 05). In the DNA migration test, there were significant differences between the spleen cells and kidney cells or between liver cells and kidney cells of the three tested groups ( P < 0 05).The percentages of cells with comets and the DNA migrations of the three types cells in tested groups were low significantly ( P <0 01, P <0 001) compared with the corresponding positive control groups. Conclusion The effects of mechanical mince on the DNA damages of spleen,liver and kidney cells became stronger and stronger orderly. Spleen and liver can be dissociated into single cells by mechanical mince more appropriately.