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目的探索不同剂量亚砷酸钠暴露不同时间对诱导人永生化角质形成(HaCaT)细胞活性氧(ROS)产生的影响以及低剂量亚砷酸钠预处理HaCaT细胞对诱导ROS水平改变的时间效应。方法设未预处理组[分别加入终浓度为0(对照)、0.15、0.6、2.5、10μmol/L的亚砷酸钠染毒8、24、72 h]和预处理8 h组(加入0.15μmol/L亚砷酸钠预处理8 h后,加入10μmol/L亚砷酸钠染毒8 h)及预处理24 h组(加入0.15μmol/L亚砷酸钠预处理24 h后,加入10μmol/L亚砷酸钠染毒8 h)。利用2’,7’-二乙酰二氯荧光素(DCFH-DA)通过流式细胞仪检测细胞内ROS水平。结果与对照组比较,各浓度亚砷酸钠染毒8 h和0.6、2.5、10μmol/L亚砷酸钠染毒24 h及0.6、2.5μmol/L亚砷酸钠染毒72 h后HaCaT细胞内ROS水平均较高,而0.15、10μmol/L亚砷酸钠染毒72 h后HaCaT细胞内ROS水平均较低,差异有统计学意义(P<0.05)。亚砷酸钠染毒8 h时,HaCaT细胞内ROS水平达到峰值;之后,低剂量(0.15、0.6μmol/L)亚砷酸钠染毒组HaCaT细胞内ROS水平于24 h下降,72 h有所回升,而高剂量(≥2.5μmol/L)亚砷酸钠染毒组HaCaT细胞内ROS水平则呈持续下降。预处理8 h组HaCaT细胞内ROS水平高于预处理24 h组,差异均有统计学意义(P<0.05);与10μmol/L亚砷酸钠染毒未预处理HaCaT细胞8 h组比较,预处理8 h组HaCaT细胞内ROS水平较高,预处理24 h组HaCaT细胞内ROS水平较低,差异均有统计学意义(P<0.05)。结论 HaCaT细胞急性暴露于亚砷酸钠后能够引起HaCaT细胞内ROS产生增加,而不同剂量亚砷酸钠暴露诱导HaCaT细胞ROS水平的改变与暴露时间密切相关,且低剂量亚砷酸钠暴露对HaCaT细胞产生的适应性反应可能取决于其作用时间。
Objective To explore the effect of different doses of sodium arsenite on the induction of reactive oxygen species (ROS) production in human immortalized keratinocytes (HaCaT) cells and the time effect of low dose sodium arsenite pretreatment on HaCaT cells induced by ROS. Methods The rats in untreated group were treated with sodium arsenite (0, 0, 0, 0, 0, 0.6, 2.5 and 10 μmol / L, respectively for 8, 24 and 72 h) / L sodium arsenite pretreatment 8 h, adding 10μmol / L sodium arsenite 8 h) and pretreatment 24 h group (0.15μmol / L sodium arsenite pretreatment 24 h, 10μmol / L sodium arsenite exposure 8 h). Intracellular ROS levels were measured by flow cytometry using 2 ’, 7’-diacetyl dichlorofluorescein (DCFH-DA). Results Compared with the control group, HaCaT cells were treated with sodium arsenite at various concentrations for 8 h and at 0.6, 2.5 and 10 μmol / L for 24 h and arsenite at 0.6 and 2.5 μmol / L for 72 h, respectively ROS levels were higher in HaCaT cells treated with 0.15,10μmol / L sodium arsenite for 72 h, the difference was statistically significant (P <0.05). At 8 h after arsenite treatment, the level of ROS in HaCaT cells peaked. After that, the intracellular ROS levels in HaCaT cells treated with sodium arsenite at a low dose (0.15, 0.6 μmol / L) decreased at 24 h, and at 72 h While the level of ROS in HaCaT cells treated with sodium arsenite at a high dose (≥2.5μmol / L) continued to decline. The level of ROS in HaCaT cells pretreated for 8 h was significantly higher than that in HaCaT cells treated with 10 μmol / L sodium arsenite for 8 h (P <0.05) The level of ROS in HaCaT cells pretreated for 8 h was higher than that in HaCaT cells pretreated for 8 h, and the level of ROS was lower in HaCaT cells pretreated for 8 h (P <0.05). Conclusions The acute exposure of HaCaT cells to arsenite causes the increase of ROS production in HaCaT cells. However, the change of ROS level induced by different doses of sodium arsenite exposure is closely related to the exposure time, and the exposure of low dose sodium arsenite The adaptive response produced by HaCaT cells may depend on their duration of action.