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目的探讨缺氧时肝癌细胞SMMC-7721中乏氧诱导因子-1α(HIF-1α)、己糖激酶II(HKII)表达及对细胞周期调控机制的影响。方法人肝癌细胞SMMC-7721分成3组:缺氧12h组、缺氧24h组及对照组。缺氧组分别在缺氧条件下(37℃,5%CO2、2%O2饱和度)培养12h和24h,对照组置于正常氧浓度条件下(37℃,5%CO2、21%O2饱和度)培养24h。流式细胞仪检测细胞周期分布,免疫组织化学SP法检测HIF-1α表达,荧光定量PCR法检测HKIImRNA表达量。结果缺氧状态下细胞周期分析处于G0/G1期细胞百分比明显增多,处于G2/M期的细胞减少,并且可见到较明显的凋亡峰的形成,缺氧12h组、缺氧24h组的G0/G1期比例显著高于对照组(P<0.05);HIF-1α和HKII在两组缺氧组与对照组比较,其表达量明显增加(P<0.05),缺氧24h组与缺氧12h组比较差异有统计学意义(P<0.05)。结论缺氧导致细胞阻滞在G0/G1期,HIF-1α可刺激人肝癌细胞HKII表达的增加,以HIF-1α为药物靶点可望成为治疗肝癌的重要新方法。
Objective To investigate the effects of hypoxia inducible factor-1α (HIF-1α) and hexokinase II (HKII) on cell cycle regulation in hepatocellular carcinoma SMMC-7721 cells under hypoxia. Methods Human hepatocellular carcinoma cells SMMC-7721 were divided into 3 groups: hypoxia 12h group, hypoxia 24h group and control group. The hypoxia group was cultured under hypoxic conditions (37 ℃, 5% CO2, 2% O2 saturation) for 12h and 24h, respectively. The control group was exposed to normal oxygen concentration (37 ℃, 5% CO2, 21% O2 saturation ) For 24 h. The cell cycle distribution was detected by flow cytometry, the expression of HIF-1α was detected by immunohistochemical SP method, and the expression of HKII mRNA was detected by fluorescence quantitative PCR. Results The percentage of cells in G0 / G1 phase in hypoxia group increased significantly, the cells in G2 / M phase decreased, and the more obvious peak of apoptosis was observed. In hypoxia 12h group and G0 / G1 phase was significantly higher than that of the control group (P <0.05). Compared with the control group, the expression of HIF-1α and HKII in the hypoxia group was significantly increased (P <0.05) The difference was statistically significant (P <0.05). Conclusion Hypoxia leads to cell arrest in G0 / G1 phase. HIF-1α can stimulate the expression of HKII in human hepatocellular carcinoma cells. HIF-1α is an important drug target for hepatocellular carcinoma.