葡萄糖浓度波动对人外周血内皮祖细胞增殖、凋亡及内皮型一氧化氮合酶活性的影响

Effects of glucose concentration fluctuation on proliferation,apoptosis and activity of endothelial nitric oxide synthase of endothelial progenitor cells

目的研究葡萄糖浓度波动对人外周血内皮祖细胞(EPCs)增殖、凋亡及内皮型一氧化氮合酶(eNOS)活性的影响。方法采用密度梯度离心法分离外周血单个核细胞,经含血管内皮生长因子、碱性成纤维细胞生长因子和10%胎牛血清的M199培养基培养7d,贴壁细胞鉴定为EPCs。细胞同化后给予葡萄糖浓度5.5mmol/L(正常对照组)、30mmol/L(糖浓度固定组)和20或40mmol/L(平均浓度为30mmol/L,波动间期为3h,糖浓度波动组)。干预6、12、24、48、96h后,采用WST-1细胞增殖检测试剂盒处理细胞,酶标仪检测细胞增殖;以AnnexinV-FITC凋亡检测试剂盒处理细胞,流式细胞仪检测细胞凋亡;以一氧化氮合酶检测试剂盒处理细胞,荧光酶标仪检测eNOS活性。结果当平均浓度相同时,与糖浓度固定组相比,糖浓度波动组抑制EPCs增殖及eNOS活性的作用显著增强(P值均<0.05),凋亡率显著增高(P<0.05);糖浓度固定组与糖浓度波动组的增殖率、凋亡率、eNOS活性的差值与干预时间呈正相关(r值分别=0.937、0.927、和0.951,P值均<0.05)。结论葡萄糖浓度波动作为独立因素促进EPCs的凋亡、抑制EPCs的增殖并降低eNOS的活性。EPCs对葡萄糖浓度波动的适应性差。

Objective To observe the effects of glucose concentration fluctuations on proliferation, apoptosis and activity of endothelial nitric oxide synthase （eNOS） of endothelial progenitor cells（EPOs） derived from human peripheral blood. Methods Peripheral blood mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation. After cultured for 7 days in M199 medium containing rh-VEGF （30 ng/mL）, rh-b-FGF （6 ng/mL） and 10% Fetal Bovine Serum, the attached cells were identified and verified to be EPCs. The ceils were synchronized and then stimulated with glucose （5.5 mmol/L [normal control group], 30 mmol/L [fixed concentration group], and 20 or 40 mmol/L with a mean of 30 mmol/L and a fluctuation interval of 3 h [fluctuation group]） for different time periods （6, 12, 24, 48 and 96 h）. The proliferation of cells was examined by Microplate Spectrophotometer, the rate of apoptosis by Flow Oytometry, and the activity of eNOS by Luminescence Microplate Spectrophotometer. Results Compared with the fixed concentration group, the rate of proliferation and activity of eNOS in fluctuation group were significantly decreased （P〈 0.05）, the rate of apoptosis in the fluctuation group was significantly higher. The rates of proliferation and apoptosis and the differences of eNOS activity were correlated with the stimulation time periods in both fixed and fluctuation groups （ rprcliferation = 0. 937, rapoptosis = 0. 927, reNOS = 0. 951, all P〈0. 05）. Conclusion Glucose concentration fluctuation as an independent factor can promote the apoptosis of EPCs, depress the proliferation of EPC, and inhibit the activity of eNOS. EPCs have a poor adaptability.