Effects of glucose concentration fluctuations on oxidative stress in peripheral blood endothelial progenitor cells

AIM To observe the effect of glucose concentration fluctuations on the active oxygen level in peripheral blood endothelial progenitor cells (EPCs). METHODS Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation. After the cells were cultured for 7 days, the cells adhering to culture dish were characterized by FITC-UEA-I and DiI-acLDL double staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of CD34, CD133 and VEGFR-2 with flow cytometry. Several groups of attached cells were incubated with glucose in a series of fluctuant amplitudes (0, 10 and 20 mmol/L), fluctuant intervals (3, 6 and 12 h) and mean concentrations (20, 25 and 30 mmol/L) for different durations (6, 12, 24, 48 and 96 h). A probe, chloromethyl derivative of dichlorodihydrofluorescein diacetate (CM-H2DCFDA) used to evaluate intracellular reactive oxidative species production, was incubated with EPCs for 20 minutes. The mean fluorescence intensity was determined in alive EPCs with flow cytometry. RESULTS Intracellular mean fluorescence intensity was positively proportional to fluctuant amplitude of glucose concentration and average concentration of glucose (P<0.05). The smaller the fluctuant interval of glucose concentration was, the higher was the mean fluorescence intensity (P<0.05). With the passage of time, the influence of average concentration of glucose on the intracellular mean fluorescence intensity gradually decreased and the influence of glucose fluctuations gradually increased (P<0.05). CONCLUSION Concentration of glucose fluctuations plays an important role in the chronic oxidative stress produced by glucose in EPCs of human peripheral blood.