铅对原代培养海马神经细胞DNA损伤作用

Effect of lead exposune on DNA damage of primarily cultured rat hippocampal neurons

目的通过检测细胞生长抑制和凋亡相关的DNA损伤蛋白在大鼠原代培养海马细胞中的表达与原代培养海马细胞DNA损伤的相关性,探讨铅的神经毒理作用机制。方法原代培养的海马神经细胞,于培养7 d全量更换培养液后加入不同浓度醋酸铅0.2、1.0、10μmol/L,置于37℃、5%CO2培养箱继续培养24 h;蛋白免疫印迹(west-ern blot)方法检测生长抑制DNA损伤诱导因子(GADD 153)表达;彗星实验检测大鼠海马原代培养神经细胞DNA损伤程度。结果与对照组比较,随着染铅浓度增高,原代培养的海马神经细胞中的DNA的尾距(OTM)和彗尾值增大,0.2、1.0μmol/L染铅组GADD 153蛋白表达较弱,染铅浓度为10μmol/L时,表达明显增加。结论细胞DNA损伤与GADD 153蛋白表达呈一定相关性。

Objective To investigate the effects of different dosage lead exposure on DNA damage,the expression of growth arrest and DNA damage inducible gene 153（GADD153） in primarily cultured rat hippocampal neurons.Methods Wistar rat hippocampal neurons were primarily cultured for 7 days.Neurons in lead acetate groups were exposed to 0.2,1.0,and 10 μmol/L lead acetate.Subsequently,hippocampal neurons in each group were cultured for 24 hours.The effects of lead on DNA damage was measured with comet assay,and GADD153 expression was measured using western blot analysis.Results Olive tail moment in all lead-treated groups and the percentage of DNA in the tail in 1.0 μmol/L and 10 μmol/L lead acetate groups were significantly increased compared with those of the control group（P0.05）.In addition,the percentage of tail DNA was higher in the 0.2 μmol/L lead acetate group compared with that of the control group（P0.05）.After incubation with 0.2,1.0,and 10 μmol/L lead acetate for 24 hours,GADD153 expression gradually decreased with the increase of lead acetate concentration（1.0-10 μmol/L）.Conclusion Lead exposure to primarily cultured rat hippocampal neurons results in dose-dependent disturbed cellular homeostasis,including DNA damage and reduced GADD153 expression.