低浓度葡萄糖诱导ECV304细胞损伤机制的初探

Reactive oxygen species participates in low glucose-induced injury of ECV304 cells

目的探讨低浓度葡萄糖对人脐静脉内皮细胞株ECV304细胞损伤的可能机制。方法以人脐静脉内皮细胞株ECV304为细胞模型,用低浓度葡萄糖刺激ECV304细胞,流式细胞仪测定12h内不同时间点细胞ROS产量,之后用低浓度葡萄糖及NADPH氧化酶抑制剂apocynin联合刺激ECV304细胞12h,用流式细胞仪测定ROS产量,MTT法测定细胞活力,光泽精化学发光法测定NADPH氧化酶活性。结果低浓度葡萄糖能够显著增加ECV304细胞ROS产量,ROS产量具有时间依赖性和葡萄糖浓度依赖性,低糖能增加NADPH氧化酶的活性。用NADPH氧化酶抑制剂apocynin预处理细胞,能使2.8mmol/L组活性氧产量降低44%,细胞活力提高32%,0mmol/L组活性氧产量降低60%。结论低糖可以显著增高ECV304细胞氧化应激水平而参与内皮细胞损伤,其部分机制可能与NADPH氧化酶激活有关。

Objective To investigate the mechanism of low glucose-induced injury in human ECV304 cells.Methods Human umbilicalvein endothelial cell line ECV304 were stimulated with low concentrations of glucose.The level of reactive oxygen species（ROS） in cells was detected at different time points within 12 h by kinetic measurement of dichlorofluoroscein（DCF） fluorescence produced by oxidation of an oxidant-sensitive dye 2,7-dichlorefluorescin（DCFH）.ECV304 cell viability was assessed with MTT assay and NADPH oxidase activity detected using lucigenin-enhanced chemiluminescence assay following cell stimulation with low glucose and apocynin.Results Low-glucose exposure of ECV304 cells time-and dose-dependently induced ROS production,and which was decreased by apocynin treatment.Apocynin pretreatment of the cells inhibited ROS production by 44% in cells exposed to 2.8 mmol/L glucose and by 60% in cells without glucose exposure.Conclusion Low glucose of ECV304 cells induces ROS production to cause cell injury,which is mediated partially by NADPH oxidase activation.