缺氧对肠上皮细胞缺氧诱导因子1α活化影响的实验研究

Effect of hypoxia on hypoxia inducible factor let（HIF-1α） activation in intestinal epithelial cells

目的了解缺氧对肠上皮细胞缺氧诱导因子1α（HIF-1α）活化的影响。方法将肠上皮细胞分为常氧处理（正常对照）、缺氧（设缺氧1、2、6、12、24h）及缺氧＋寡霉素处理（分别用浓度为5、10、20、40μg／mL寡霉素处理1h，再缺氧6h）。采用蛋白质印迹法检测HIF-1α蛋白表达，免疫荧光法观察HIF-1α向细胞核转位的情况。结果与正常对照（0．08±0．07）相比较，缺氧1h肠上皮细胞HIF-1α蛋白表达（0．52±0．30）即显著升高（P〈0．05），6h达峰值（2．37±1．08，P〈0．05），同时HIF-1α向细胞核转位也明显增加。寡霉素呈剂量依赖性地抑制缺氧引起的肠上皮细胞HIF-1α蛋白表达增加，用5、10、20及40μg／mL寡霉素处理的缺氧肠上皮细胞HIF-1α蛋白表达量分别为1．62±0．96、1．48±0．56、1．08±0．36及0．58±0．11，均较单纯缺氧6h（2．67±1．38）显著降低（P〈0．05），HIF-1α向细胞核转位也被抑制。结论呼吸链抑制剂寡霉素可抑制缺氧肠上皮细胞HIF-1α活化，线粒体呼吸链可能在其发生机制中具有重要作用。

Objective To investigate the effect of hypoxia on HIF-1α activation in intestinal epithelial cells. Methods Intestinal epithelial ceils were randomly divided into normal control group, hypoxia group and hypoxia plus oligomycin group （oligomycin group）. In hypoxia group, the cells were exposed to hypoxia for 1, 2, 6, 12 and 24 h. In oligomycin group, the cells were treated with oligomyein in concentration of 5, 10, 20 and 40 μg/mL for 1 h prior to 6-hour hypoxic exposure. HIF-1α protein expression was assayed by western blot method. Nuclear translocation of HIF-1α was detected by immunofluorescence analysis. Results Compared with that in control group（0.08±0.07） , HIF-1α protein expression in hypoxia group increased significantly at 1 h （0.52±0.30, P 〈0.05） , and reached the peak value （2.37±1.08, P 〈 0. 05） at 6 h. Nuclear translocation of HIF-1α was also induced by hypoxia. HIF-1α protein expression in oligomycin group in the concentration of 5, 10, 20 and 40 μg/mL of oligomycin was 1.62±0.96, 1.48±0. 56, 1.08±0.36 and 0.58±0.11 respectively, which was significantly lower than that only after exposure to hypoxia for 6h（2.67±1.38, P〈0.05）. The nuclear translocation of HIF-1α induced by hypoxia was also obviously inhibited by oligomycin pretreatment. Conclusion Oligomycin, a specific inhibitor of respiratory chain, inhibits HIF-1α activation, which suggests that mitochondria respiratory chain may play an important role in aforementioned process.