低氧诱发人肝癌细胞株HepG2中IAP-2表达及相关机制  

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

作　　者：谷华[1] 赵秋[1] 廖家智[1] 杜静[1] 覃华[1] 刘南植[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院消化内科,武汉430030

出　　处：《华中科技大学学报（医学版）》2006年第3期310-313,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：湖北省科技攻关计划资助项目(No.2002AA304B05)

摘　　要：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),35、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4 h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。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.

关 键 词：低氧 肝癌细胞 凋亡抑制蛋白2 

分 类 号：R735.7[医药卫生—肿瘤]