低氧诱发人肝癌细胞株HepG2中IAP-2表达及相关机制

目的 研究极度低氧下人肝癌细胞株HepG2中凋亡抑制蛋白2（IAP-2）表达的变化，初步探讨其与缺氧诱导因子1（HIF-1）的相关性。方法 HepG2细胞株分组孵育，用免疫细胞化学定性检测IAP-2蛋白，再用Western blot半定量检测IAP-2蛋白变化，同时对比HIF-1蛋白表达；用RT-PCR检测IAP-2基因水平改变。结果 免疫细胞化学检测IAP-2蛋白在HepG2细胞中呈阳性表达，定位于胞质。Western blot显示，极度低氧1 h IAP-2蛋白表达明显高于常氧组（t=5．723，P〈0．05），3、5 h IAP-2持续高表达，复氧后恢复基线水平；实验还显示HIF-1和IAP-2的蛋白表达具有差异性：常氧组HIF-1未表达，低氧4h可诱发，IAP-2保持基线水平；极度低氧HIF-1无变化，IAP-2表达明显增高；复氧后HIF-1不表达，IAP-2恢复基线表达；加入氯化钴后HIF-1恢复表达，IAP-2保持基线。RT-PCR检测表明，极度低氧1 h IAP-2 mRNA表达明显高于常氧组（t=7．097，P〈0．05），3、5 h IAP-2持续高表达，该结果与上述IAP-2蛋白表达具有一致性。结论 首次表明极度低氧下IAP-2在人肝癌细胞株HepG2表达上调，并具有独立于HIF-1的抗凋亡机制。

Expression and Related Mechanism of Apoptosis Inhibitory Protein 2 in Hepatic Cancer Cell HepG2 Induced by Hypoxia

Objective To investigate the changes in the expression of apoptosis inhibitory protein 2(IAP-2) in hepatic cancer cell HepG2 under severe hypoxia and explore its relation with hypoxia inducible factor 1(HIF-1).Methods HepG2 cells were cultured in different groups.Immunocytochemistry was used to qualitatively assay IAP-2 protein.Western blot analysis was applied to semi-quantitatively determine IAP-2 protein and HIF-1 protein.RT-PCR was performed to detect the mRNA expression of IAP-2.Results The immunocytochemical staining showed the positive expression of IAP-2 protein in HepG2 cytoplasma.Western blot analysis revealed the expression of IAP-2 protein could be markedly induced(t=5.723,P<0.05) under severe hypoxia for 1 h and remain high after 3 or 5 h.There was difference in the expression of IAP-2 and HIF-1 proteins.HIF-1 was undetectable in normoxic HepG2 cells,but induced by hypoxia.Under sever hypoxia HIF-1 was modest but IAP-2 was abundantly detected.After re-oxygenation HIF-1 was degraded and IAP-2 returned to basal level.Colalt chloride activated HIF-1 but not IAP-2.RT-PCR analysis found the expression of IAP-2 mRNA after 1 h of sever hypoxia was evidently higher than that in normoxia(t=7.097,P<0.05),and remained high after 3 or 5 h.Conclusion Severe hypoxia induced the up-regulation of IAP-2 in HepG2 through HIF-1-independent pathways.