【作者】 桑丹； 薛耀明； 沙建平； 高方； 周琳； 符霞军； 李际敏；

【机构】 南方医科大学南方医院；

【摘要】 目的:观察体外持续高葡萄糖浓度和葡萄糖浓度波动条件下,用N-甲酰基-甲硫氨酰基-亮氨酰基苯丙氨酸(fMLP)激活正常人外周血白细胞(WBCs),观察细胞内还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)水平及活性氧(ROS)产量,探讨两种条件对白细胞呼吸爆发的影响。方法:以体外培养正常人外周血WBCs为研究对象,实验分为正常对照组(5.5mmol/L D-葡萄糖)、持续高糖组(16.7mmol/L D-葡萄糖)与波动组(5.5-16.7 mmol/L D-葡萄糖交替),所有组处理24h后以fMLP干预30min激活WBCs。紫外分光光度计在340nm处测细胞裂解上清液吸光值,计算WBCs内NADPH含量;用二氯荧光素双醋酸盐(2’,7’-H2 DCFH-DA)作为荧光探针,流式细胞仪检测WBCs产生ROS的水平。结果:与正常对照组相比,持续高糖和波动高糖均能使经fMLP刺激后WBCs的NADPH[分别为0.098±0.015,0.064±0.010和0.033±0.003(μmol/mg)]和ROS水平(分别为3269.425±270.678,2075.322±334.372和1190.537±155.225)降低(P<0.05),且波动组作用更明显(P<0.05)。结论:持续性高葡萄糖浓度和波动性高葡萄糖浓度均能使人外周血WBCs内NADPH水平及ROS产生能力下降,波动性高葡萄糖浓度效果更加明显,导致WBCs呼吸爆发功能受损。更多还原

【Abstract】 Objective:Our study aims to investigate the effects of continuing high glucose and fluctuating high glucose on reduced form of nicotinamide-adenine dinucleotide phosphate(NADPH) level and the production of reactive oxygen species(ROS) of healthy human peripheral white blood cells(WBCs) after N-formyl-methionyl-leucylphenylalanine(fMLP) intervention and respiratory burst ability of WBCs.Methods:Isolated healthy human peripheral WBCs were incubated in the medium respectively containing normal glucose(5.5mmol/L D-Glu),continuing high glucose(16.7mmol/L D-Glu) and fluctuating high glucose(5.5mmol/L and 16.7mmol/L D-Glu alternatively) for 24 h,then with fMLP intervention for 30 min to stimulate WBCs.Intracellular NADPH contents was assayed by measuring the absorbance of cell supernatant at 340 nm on spectrophotometer.Intracellular ROS was evaluated by the staining fluorescent probe of 2’,7’-dichlorofluoresce in diacetate on flow cytometry.Results:Compared with the normal control group,NADPH contents and production of ROS of the cells were both found decreased in continuing high glucose and fluctuating high glucose after fMLP intervention(P<0.05)and fluctuating high glucose had more obvious effects(P<0.05).Conclusion:Healthy human peripheral WBCs exposed to continuing high glucose and fluctuating high glucose concentration both have decreased NADPH contents and the ability of ROS production,and the latter had more obvious effects leading to the impairment of respiratory burst of WBCs.更多还原