低氧对人支气管上皮细胞HSP70和HIF-1α表达的影响

Effect of hypoxia on the expression of HSP70 and HIF-1α in HBE cells

将人支气管上皮细胞(HBE)在低糖 DMEM 培养基、1%O_2培养不同时间后,分别用RT-PCR 和 Western blot 方法检测细胞 HSP70、HIF-1α的 mRNA 和蛋白表达水平,同时采用免疫组化技术观察低氧对 HSP70蛋白的影响.对照组培养于低糖、常氧条件下.结果表明,低氧条件下,HBE 细胞 HSP70 mRNA 和蛋白表达都有时间依赖性.在1h 时 HSP70mRNA转录水平即开始增加,与对照相比差异显著(P<0.05);在12h 时转录水平最高;而 HSP70蛋白的表达略有滞后,在3h 差异显著(P<0.05).低氧诱导因子 HIF-1αmRNA 水平在1h 时显著升高,在6h 和12h 时与对照相比差异极显著(P<0.01);HIF-1α蛋白的表达也同步增加,在6h 时达到峰值.这为慢性阻塞性肺疾病(COPD)的发病机制研究提供了一种可供选择的细胞模型.

Serum-deprived cells were maintained for the indicated times in DMEM （low glucose ）culture medium, under 1% oxygen. Total RNA and protein were isolated from each group and subjected to RT-PCR and western blot analysis for expression of HSP70 and HIF-1α, respectively. Immunohistochemical technique was applied to observe the expression of HSP70 induced by 1% oxygen. Control group was exposed to normoxia. It was shown that hypoxia induced the expressions of HSP70 mRNA and protein in a time-dependent manner. RT-PCR analysis revealed that the amount of HSP70 mRNA in HBE ceils exposed to 1% oxygen significantly increased at 1 h（P〈0.05）compared with that in cells exposed to normoxia. While HSP70 protein displayed significant up-regulation at 3 h. The induction of HSP70 protein and mRNA both reached highest at 12 h （P 〈0.01 versus the control） then decreased. The effect of hypoxia on the expression of HIF-1αmRNA was also time dependent with the increases being significant and maximal at 1 h and 12 h. HIF-1αprotein also increased significantly in hypoxic HBE cells at 1 h （P〈0.05）as compared to the control and reached maximally at 6 h. These results provide some useful information for the study of COPD.