葡萄糖浓度波动对人外周血内皮祖细胞氧化应激的影响

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

目的观察葡萄糖浓度波动对人外周血内皮祖细胞(EPCs)活性氧水平的影响。方法采用密度梯度离心法分离外周血单个核细胞,培养7d后,收集贴壁细胞,进行FITC-UEA-Ⅰ、DiI-acLDL双染色鉴定,并用流式细胞术检测细胞的表面标志CD34、CD133和血管内皮生长因子受体-2。EPCs给予不同浓度波动幅度(0、10、20mmol/L)、不同浓度波动间期(3、6、12h)、不同平均浓度(20、25、30mmol/L)的葡萄糖,培养一定时间(6、12、24、48、96h)后,以氯甲基二氯二氢荧光素二乙酯染色细胞,用流式细胞术检测细胞内平均荧光强度(MFI)。结果葡萄糖浓度波动幅度越大、浓度波动间期越小、平均浓度越高,细胞内MFI越高(P<0.05)。平均浓度对MFI的影响随时间减小;而浓度波动对MFI的影响随时间增大(P<0.05)。结论葡萄糖浓度波动在葡萄糖所致人外周血EPCs慢性氧化应激中起重要作用。

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） , fluetuant 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 fluetuant 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.