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目的探讨缺氧对肿瘤细胞周期调控的机制及乏氧诱导因子-1α(HIF-1α)、己糖激酶Ⅱ(HKⅡ)表达的影响。方法人肝癌细胞HepG2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、2.0%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、21.0%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S2P法检测HIF-1α表达,荧光定量PCR法检测HKⅡmRNA表达量。结果缺氧情况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组比较有显著性差异(p<0.05)。结论HIF-1α可刺激人肝癌细胞HKⅡ表达的增加,细胞阻滞在G0/G1期,以HIF-1α为药物靶点可望成为治疗肝癌基因治疗的新途径。
Objective To investigate the mechanism of hypoxia on the regulation of tumor cell cycle, and the effects of hypoxia-inducible factor-1α (HIF-1α) and hexokinase II (HKII) expression. Methods HepG2 cells were divided into 3 groups: hypoxia 12 h group, hypoxia 24 h group and control group. The hypoxic groups were incubated under hypoxic conditions (37°C, 5% CO2, 2.0% O2 saturation) for 12 h and 24 h, and the control group was exposed to normal oxygen concentrations (37°C, 5% CO2, 21.0% O2 saturation). ) Culture 24h. Flow cytometry was used to detect the distribution of cell cycle. Immunohistochemistry was used to detect the expression of HIF-1α. Fluorescent quantitative PCR was used to detect the expression of HKII mRNA. RESULTS: Under hypoxia, the percentage of cells in G0/G1 phase was significantly increased, cells in G2/M phase decreased, and more obvious apoptotic peaks were seen. G0 in hypoxia 12 h group and hypoxia 24 h group were observed. The proportion of /G1 phase was significantly higher than that of the control group (p<0.05); the expression of HIF-1α and HKII in the hypoxic group was significantly higher than that in the control group (p<0.05), and the hypoxia 24 h group was associated with hypoxia 12 h. There was a significant difference between the groups (p<0.05). Conclusion HIF-1α can stimulate the increase of the expression of HK II in human hepatoma cells. The cell arrest is in G0/G1 phase. HIF-1α as a drug target is expected to be a new approach for gene therapy of liver cancer.