【摘要】 目的探讨不同间歇低氧（IH）和持续低氧（CH）模式下内皮细胞的炎性损伤。方法在细胞培养舱中程控产生预置的间歇低氧/再氧合（IH/ROX）循环环境,将人脐静脉内皮细胞可传代细胞株 ECV304暴露于该环境。分为间歇正常氧（IN）组（21%O₂ 15 s/21% O₂ 225 s,60次循环）,IH组（1.5%O₂ 15 s/21% O₂ 225 s,30、60次循环）,IH 高碳酸组（1.5%O₂ 20%CO₂ 15 s/21% O₂ 5%CO₂ 225 s,60次循环）,CH 组（1.5%、10% O₂,持续时间为15、30、60 min）,CH 高碳酸组（10% O₂10%CO₂,持续时间为15、30、60 min）,CH 累加 IH 组（1.5% O₂ 15 s/10% O₂ 225 s,60次循环）,不同IH 程度组（1.5%、10% O₂ 15 s/21% O₂ 225 s,60次循环）,不同 IH 频率组（1.5%O₂ 15 s/21%O₂225 s、315 s、495 s、105 s,60次循环）和不同 IH 时限组（1.5% O₂ 15 s、30 s/21% O₂ 225 s,60次循环）。而后采用酶联免疫吸附法（ELISA）测定培养基中白细胞介素6（IL-6）和肿瘤坏死因子α（TNF-α）的水平,并测定细胞裂解液总蛋白含量进行标化。结果 IN 组 IL-6和 TNF-α水平为（374.06±38.10）和（31.96±13.64）pg·ml^-1·100 mg 蛋白^-1,而 IH 组2的 IL-6和 TNF-α水平为（770.40±21.60）和（126.93±2.58）pg·ml^-1·100 mg 蛋白^-1,明显高于 IN 组（均 U=0.000,P=0.002）;IH 高碳酸组 IL-6水平为（829.27±7.16）pg·ml^-1·100 mg 蛋白^-1,高于 IH 组2（U=0.000,P=0.002）。CH 高碳酸组的 IL-6均明显高于相同经时反应进程的 CH 组（均 U=0.000,P=0.002）,CH 累加 IH 组 IL-6和 TNF-α水平为（536.74±14.97）和（51.10±6.80）pg·ml^-1·100 mg蛋白^-1,高于 IN 组却低于 IH 组2（均 x²=23.4,P<0.05）。IL₆和 TNF-α水平随 IH 程度加重而增加（均 x²=23.4,P<0.05）,不同 IH 频率组的 IL-6和 TNF-α水平则呈现出复杂变化。结论 IH 和 CH对内皮细胞造成明显炎性损伤并具有程度依赖性,伴高碳酸暴露时损伤更重;IH/ROX 对内皮细胞的炎性损伤来源于 ROX 时段而非来源于 IH 时段。更多还原

【Abstract】 Objective To investigate the damage of different patterns oi intermittent hypoxia（IH） and continuous hypoxia（CH）on endothelial cells.Methods Human umbilical vein endothelial cells of the line ECV304 were cultured in a program-controlled gas delivery system newly developed and divided into 8 groups to undergo different IH/reoxygenation（ROX）cycles so as to simulate the patterns of hypoxic episode seen in recurrent apnea and chronic obstructive pulmonary disease:intermittent normoxia（IN）group （exposed to 21% O₂ 15 s/21% O₂ 225 s for 60 cycles）,IH group（exposed to 1.5% O₂ 15 s/21% O₂ 225 s for 30 or 60 cycles）,IH hypercapnia group（exposed to 1.5% O₂ and 20% CO₂ 15 s/21% O₂ and 5% CO₂ 225 s,for60 cycles）,continuous hypoxia（CH group,exposed to 1.5% or 10% O₂ for 15,30 or60 min）, CH hypercapnia group（exposed to 10% O₂ and 10% CO₂ for 15,30 or 60 min）,CH added to IH group （exposed to 1.5% O₂ 15 s/10% O₂ 225 s for 60 cycles）,different intermittent hypoxia extent group （exposed to 1.5% or 10% O₂ 15 s/21% O₂ 225 s for 60 cycles）,different intermittent hypoxia frequency group（exposed to 1.5% O₂ 15 s/21% O₂ 225 s 315 s,495 s or 105 s for 60 cycles）,and different intermittent hypoxia duration group（exposed to 1.5% O₂ 15 s or 30 s/21% O₂ 225 s for 60 cycles）.Then ELISA was conducted to examine the levels of interleukin-6（IL-6）and tumor necrosis factor-α（TNFα） Bicinchoninic acid method was used to standardize the cell total protein level.Results The levels of IL-6 and TNFα levels in the IH group were（770.40±21.60）and（126.93±2.58）pg·ml^-1·100 mg protein^-1respectively,both significantly higher than those in the IN group [（374.06±38.10）and （51.96±13.64）pg·ml^-1·100 mg protein^-1respectively,U=0.000,P=0.002],but significantly lower than those in the IH hypercapnia group[（829.27±7.16）and（78.77±4.00）pg·ml^-1·100 mg protein^-1respectively,U=0.000,P=0.002].The IL-6 levels of the CH hypercapnia 15,30,and 60 min subgroups were all significantly higher than those of the corresponding CH subgroup（U=0.000,P= 0.002）.The IL-6 and TNFα levels of the CH added to IH group were（536.74±14.97）and（51.10± 6.80）pg·ml^-1·100 mg protein^-1respectively,both were significantly higher than those of the IN group, but significantly lower than those of the IH group（X²=23.4,P<0.05）.The levels of IL-6 and TNFα increased along with the increase of the IH degree（X²=23.4,P<0.05）.The level changes of IL-6 and TNFα of the groups with different intermittent hypoxia frequency and with different intermittent hypoxia duration were complicated.Conclusion IH and CH significantly damage the endothelial cells dose- dependently,especially combined with hypercapnia.In IH/ROX,the inflammatory damage comes from ROX phase but not IH phase.Hypoxia duration and hypoxia frequency are also important parameters in the activation of inflammation.更多还原