低氧条件下血红素加氧酶1过表达对人肝癌细胞的保护作用  

Protective effect of HIF-1α-dependent HO-1 overexpression on hypoxic human hepatoma cells in vitro

作　　者：梁飞[1] 朱晓洁[1] 王秀宏[1] 

机构地区：[1]哈尔滨医科大学生物化学与分子生物学教研室,150086

出　　处：《中华肿瘤杂志》2009年第8期587-591,共5页Chinese Journal of Oncology

基　　金：黑龙江省自然科学基金（D200609）;黑龙江省博士后启动基金

摘　　要：目的探讨低氧条件下人肝癌细胞（HepG2）中血红素加氧酶1（HO-1）过表达对细胞的保护作用，及其与低氧诱导因子1α（HIF-1α）的关系。方法应用低氧气体（0．5％O2、94．5％N2、5％CO2）和化学模拟低氧（250μmol／LCoCl2）方法刺激HepG2细胞。采用逆转录聚合酶链反应（RT—PCR）方法，从mRNA水平上检测低氧条件下HO-1和HIF-1α的表达。采用Western bolt方法，从蛋白水平上检测低氧条件下HO-1和HIF-1α的表达及其相关性。采用四甲基偶氮唑蓝（MTF）方法，分析低氧刺激后细胞的存活率，以及低氧刺激同时抑制HO-1蛋白表达后细胞的存活率。采用超氧化物歧化酶（SOD）试剂盒，检测低氧刺激细胞后总SOD的活性，以及抑制HO-1蛋白表达后细胞中总SOD的活性。结果低氧诱导的HepG2细胞中，HO-1mRNA和蛋白过表达。用锌原卟啉IX（ZnPPIX）抑制低氧条件下HO-1蛋白过表达，可导致HepG2细胞在低氧应激条件下存活率明显降低（P〈0．01）。低氧条件下，HepG2细胞中总SOD的活性显著升高（P〈0．05）；而在抑制低氧条件下HO-1蛋白过表达后，HepG2细胞中总SOD活性明显降低（P〈0．01）。低氧条件下，HIF-1α蛋白表达显著升高，抑制HIF—1α蛋白表达后，HO-1蛋白表达明显降低；而抑制低氧条件下HO-1的过表达后，HIF-1α蛋白表达没有明显改变。结论低氧诱导HepG2细胞中HO-1蛋白的过表达依赖或部分依赖HIF—1α；HO-1的过表达对HepG2细胞抵抗低氧应激发挥保护作用；HO-1的过表达对处于低氧应激条件下细胞的保护作用可能源于SOD。Objective To investigate the protective effect of overexpressed heme oxygenase-1 （ HO- 1 ） in hypoxia and the correlation between HO-1 overexpressoin and hypoxia inducible factor-1α （HIF-1α） in human hepatoma HepG2 cells. Methods The expressions of HO-1 and HIF-1α mRNA as well as the protein were detected by RT-PCR and Western blotting, respectively. MTT assay was used to examine the relative cell survival rate. The total superoxide dismutase （SOD） activity was examined with a SOD kit. Results Hypoxia induced overexpression of HO-1 gene in HepG2 cells at transcriptional and translational levels. The relative survival rate of HepG2 cells under hypoxia was significantly decreased after the HO-1 protein overexpression was inhibited by ZnPPIX （P 〈 0.01 ）. The total SOD activity of cells was significantly increased after cells were treated by hypoxia for 16 hours （P 〈 0.05 ）, while decreased significantly by HO- 1 inhibitor ZnPPIX treatment （P 〈 0.01 ）. HIF-1α was upregnlated under hypoxia. In addition, the HO-1 overexpression under hypoxia was decreased by HIF-1α inhibitor, while the HIF-1α expression level under hypoxia was not significantly changed after HO-1 expression was inhibited by ZnPPIX. Conclusion The overexpression of HO-1 in hypoxic HepG2 cells is HIF-1α-dependent or at least partly HIF-1α-dependent. The relative survival rate of hypoxic hepatoma cells was significantly decreased by HO-1 inhibitor treatment. The results of this study may offer new thought and drug target for the therapy of human hepatoma in the future.

关 键 词：血红素加氧酶1 低氧诱导因子1Α HepG2细胞 存活率 超氧化物歧化酶 

分 类 号：R686[医药卫生—骨科学]