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目的研究不同浓度亚砷酸钠(NaAsO2)对人皮肤永生化角质形成细胞株(HaCaT)的氧化应激作用。方法用AlamarBlue还原法检测NaAsO2对细胞活力的影响,用PI染色法检测细胞的凋亡和坏死率,利用2′7′二乙酰二氯荧光素(DCFHDA)检测细胞内ROS水平,同时用荧光法测定细胞内还原型谷胱甘肽(GSH)和氧化型谷胱甘肽(GSSG)的含量。结果0001μmolL至1μmolL组的AlamarBlue还原率显著高于对照组,而10μmolL以上组的AlamarBlue还原率下降,20μmolL组的凋亡和坏死率显著高于对照组,各实验组的DCF荧光强度与对照组相比明显提高,1μmolL以上组细胞内GSH水平增高,5μmolL组GSSG水平增高。结论各浓度砷引起HaCaT细胞内ROS增多,低浓度时促进细胞增殖,高浓度则抑制细胞活力,砷诱导HaCaT细胞内的GSH增多,发挥解毒作用。
Objective To investigate the oxidative stress of human skin immortalized keratinocyte (HaCaT) cells treated with different concentrations of sodium arsenite (NaAsO2). Methods The effect of NaAsO2 on cell viability was detected by AlamarBlue reduction method. The apoptosis and necrosis of cells were detected by PI staining. The intracellular ROS level was detected by 2’7 ’diacetyl dichlorofluorescein (DCFHDA) The levels of intracellular reduced glutathione (GSH) and oxidized glutathione (GSSG) were determined. Results The reduction rate of AlamarBlue from 0001μmolL to 1μmolL group was significantly higher than that from the control group, while the reduction rate of AlamarBlue from 10μmolL group was lower than that of the control group. The apoptosis and necrosis rate of 20μmolL group was significantly higher than that of the control group. Compared with the control group, the level of GSH in the above 1μmolL group increased and the level of GSSG in 5μmolL group increased. Conclusion Arsenic can increase the intracellular ROS of HaCaT cells, increase the proliferation of HaCaT cells at low concentrations, inhibit the viability of cells at high concentrations, and increase the level of GSH in arsenite-induced HaCaT cells.