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本文研究胆固醇是否通过氧化应激机制损伤人内皮细胞DNA.实验用12.5 mg/L,25mg/L,50 mg/L胆固醇作用于人内皮细胞12 h,用免疫荧光法观察到不同浓度的胆固醇均可诱导细胞内γH2AX形成焦点,随着胆固醇浓度的增加,γH2AX焦点的荧光强度也逐渐增强.利用Western印迹及流式细胞术检测到细胞内γH2AX的量和ROS水平也随胆固醇浓度增大而增加.彗星电泳实验检测到细胞内拖尾DNA量随胆固醇浓度逐渐增加,DNA损伤加重.用抗氧化剂10 mmol/L N-乙酰半胱氨酸(NAC)预作用细胞1 h后,再用50 mg/L胆固醇作用细胞12 h,细胞内γH2AX焦点的荧光强度明显减弱,γH2AX量减少,ROS的水平下降.结果表明,胆固醇可诱导人脐静脉内皮细胞中ROS升高,导致DNA损伤.
In this study, we investigated whether cholesterol damages human endothelial cell DNA through the mechanism of oxidative stress.Experimental effects of cholesterol at concentrations of 12.5 mg / L, 25 mg / L and 50 mg / L on human endothelial cells for 12 h were observed by immunofluorescence Can induce intracellular γH2AX formation of the focus, with the increase of cholesterol concentration, γH2AX focus fluorescence intensity gradually increased.Western blot and flow cytometry detected intracellular levels of γH2AX and ROS levels also increased with increasing cholesterol concentration The amount of trailing DNA in the cells was increased by the comet assay and the DNA damage was increased with the increase of cholesterol concentration.The cells were pre-treated with 10 mmol / L N-acetylcysteine (NAC) for 1 h and then treated with 50 mg / L cholesterol-treated cells for 12 h, the fluorescence intensity of γH2AX in the cells decreased significantly, the amount ofγH2AX decreased and the ROS level decreased.The results showed that cholesterol could induce the ROS in human umbilical vein endothelial cells and lead to DNA damage.