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目的研究促红细胞生成素(erythropoietin,EPO)联合粒细胞集落刺激因子(granulocyte colony stimulating factor,G-CSF)对缺氧心肌细胞的保护作用。方法分离乳鼠心肌细胞。采用95%N2+5%CO2气体充分置换的1%胎牛血清DMEM培养液培养24h,建立缺氧心肌细胞模型。研究不同浓度的EPO和G-CSF对缺氧心肌细胞的保护作用。观察在最佳作用浓度时,EPO、G-CSF和联合用药对缺氧心肌细胞的保护作用。采用流式细胞仪检测各组心肌细胞的存活率、凋亡率和坏死率。结果缺氧心肌细胞的死亡率(26.73%)和其中凋亡细胞与总死亡细胞的比例(70.05%)明显高于正常心肌细胞(5.63%、37.83%)。EPO1U/ml浓度开始发挥对缺氧心肌细胞的保护作用,5、25和125U/ml的保护作用相似;G-CSF6ng/ml开始发挥对缺氧心肌细胞的保护作用,150ng/ml保护作用最强,30、750ng/ml保护作用相似。EPO(5U/ml)、G-CSF(30ng/ml)和联合用药[EPO(5U/ml)+G-CSF(30ng/ml)]明显减少缺氧心肌细胞的凋亡率[(8.01±2.28)%、(7.08±0.16)%、(4.57±0.99)%,与缺氧心肌细胞(18.73±1.08)%比较,P<0.01]和总死亡率[(11.88±3.69)%、(11.60±0.65)%、(8.15±3.16)%,与缺氧心肌细胞(26.73±1.30)%比较,P<0.01);EPO组与G-CSF组保护作用相近(P>0.05);联合给药组保护作用明显强于单独EPO或G-CSF给药组(P<0.01)。结论EPO和G-CSF对缺氧心肌细胞具有保护作用,5U/mlEPO和30ng/mlG-CSF是减轻缺氧心肌细胞损伤的最佳剂量。二者联合给药,保护效果优于单独用药。
Objective To study the protective effect of erythropoietin (EPO) combined with granulocyte colony stimulating factor (G-CSF) on hypoxic cardiomyocytes. Methods Isolated neonatal rat cardiomyocytes. Hypoxic cardiomyocytes were cultured in 1% fetal bovine serum (DMEM) with 95% N2 + 5% CO2 gas for 24 h. To study the protective effects of different concentrations of EPO and G-CSF on hypoxic cardiomyocytes. To observe the protective effects of EPO, G-CSF and combination therapy on hypoxic cardiomyocytes at optimal concentration. Flow cytometry was used to detect the survival rate, apoptosis rate and necrosis rate of cardiomyocytes in each group. Results The death rate of hypoxic cardiomyocytes (26.73%) and the ratio of apoptotic cells to total dead cells (70.05%) were significantly higher than that of normal cardiomyocytes (5.63%, 37.83%). EPO1U / ml concentration began to play a protective effect on hypoxic cardiomyocytes, 5,25 and 125U / ml of the protective effect is similar; G-CSF6ng / ml began to play a protective effect on hypoxic cardiomyocytes, 150ng / ml the strongest protective effect , 30,750ng / ml similar protective effect. EPO (5U / ml), G-CSF (30ng / ml) and combination therapy [EPO (5U / ml) + G-CSF (30ng / ml)] significantly reduced the apoptotic rate of hypoxic cardiomyocytes [(8.01 ± 2.28 (11.88 ± 3.69)%, (11.60 ± 0.65)%, (7.08 ± 0.16)%, (4.57 ± 0.99)%, respectively, compared with those in hypoxic cardiomyocytes (18.73 ± 1.08)%, P <0.01] ), (8.15 ± 3.16)%, compared with that of hypoxic cardiomyocytes (26.73 ± 1.30)%, P <0.01). The protective effect of EPO group was similar to that of G-CSF group Was significantly stronger than EPO alone or G-CSF administration group (P <0.01). Conclusion EPO and G-CSF have protective effects on hypoxic cardiomyocytes. 5U / ml EPO and 30ng / ml G-CSF are the best dosage to reduce the damage of hypoxic cardiomyocytes. The combination of the two, the protective effect is better than single drug.