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目的探讨缺氧-复氧对大鼠心肌微血管内皮细胞(rat myocardial microvascular endothelial cells,RMMEVCs)中缺氧诱导因子-1(hypoxia-inducible factor-1,HIF-1)表达以及钠氢交换体-1(hydrogen exchanger-1,NHE1)的表达及活性的影响。方法蛋白印迹检测缺氧培养的RMMVECs的HIF-1、NHE1蛋白水平表达,并用氯化钴模拟缺氧进行验证。进而BCECF/AM法检测细胞内pH值、酸负荷后pH回复率及对NHE1特异阻断剂DMA的反应。复氧0、24、48 h后检测缺氧影响作用的回复情况。结果缺氧导致RMMVECs中HIF-1、NHE1蛋白表达水平升高,氯化钴模拟缺氧证实NHE1表达亦升高。但缺氧致细胞内pH值下降[0 h(7.42±0.06);12 h(7.17±0.05);24 h(7.04±0.04);P<0.01],酸负荷后细胞内pH值回复率减小[(U/min)0 h(0.106±0.007);12 h(0.059±0.005);24 h(0.044±0.004);P<0.01];相对于DMA未处理组,不同缺氧时间点DMA处理组的酸负荷后pH回复率均降低[(U/min)0 h,-0.063;12 h,-0.028;24 h,-0.020]。复氧后NHE1蛋白水平逐渐下降,但复氧24 h酸负荷后细胞内pH值回复率最高(0.119 U/min),DMA抑制幅度最明显(-0.076 U/min),与0、48 h比较差异显著(P<0.01)。结论在缺氧中HIF-1升高可能导致NHE1蛋白表达升高,但NHE1活性降低,而复氧后NHE1活性升高。NHE1表达与活性改变可能是RMMVECs缺氧中pH调节的重要机制。
Objective To investigate the effects of hypoxia-reoxygenation on the expression of hypoxia-inducible factor-1 (HIF-1) in rat myocardial microvascular endothelial cells (RMMEVCs) (hydrogen exchanger-1, NHE1) expression and activity. Methods Western blotting was used to detect the expression of HIF-1 and NHE1 protein in hypoxia-cultured RMMVECs. Furthermore, BCECF / AM method was used to detect intracellular pH value, pH recovery after acid loading and response to NHE1-specific blocker DMA. After 0,24,48 h of reoxygenation, the effect of hypoxia was detected. Results Hypoxia caused the expression of HIF-1 and NHE1 protein in RMMVECs to increase, and the hypoxia of cobalt chloride confirmed that the expression of NHE1 also increased. However, the intracellular pH value decreased after hypoxia [0 h (7.42 ± 0.06), 12 h (7.17 ± 0.05), 24 h (7.04 ± 0.04), P <0.01] Compared with the DMA untreated group, the DMA treatment group at different time points of hypoxia showed no significant difference (P> 0.05); [(U / min) 0 h (0.106 ± 0.007); 12 h The acid recovery of pH decreased after [(U / min) 0 h, -0.063; 12 h, -0.028; 24 h, -0.020]. After reoxygenation, the level of NHE1 protein decreased gradually. However, the highest intracellular pH value (0.119 U / min) and the highest DMA inhibition rate (-0.076 U / min) after 24 h reoxygenation were compared with those of 0 and 48 h The difference was significant (P <0.01). Conclusion HIF-1 in hypoxia may increase the expression of NHE1 protein, but decrease the activity of NHE1, but increase the activity of NHE1 after reoxygenation. The change of NHE1 expression and activity may be an important mechanism of pH regulation in hypoxia of RMMVECs.