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目的:观察常氧酸性环境对体外肝癌细胞低氧诱导因子(hypoxia-inducible factor,HIF)-1α蛋白表达及其活性的影响,初步探讨其在肿瘤生长中的作用。方法:体外培养肝癌细胞HepG2,以MTT法测定常氧酸性环境(AP组,pH6.5)作用20h后细胞存活率,并与正常培养细胞(SD组,pH7.2)作比较;Western印迹法检测AP组和SD组HepG2细胞核内HIF-1α蛋白表达的变化;应用转录因子DNA结合ELISA试剂盒(TransAMTM)分析AP组和SD组HepG2细胞核内HIF-1DNA结合活性的变化;免疫细胞化学法和Western印迹法检测各组HepG2细胞内血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白表达的变化。结果:常氧酸性环境(pH6.5)作用20h后,AP组细胞存活率为(98.71±1.79)%,与SD组相比轻微降低,但无统计学意义(P>0.05)。Western印迹结果表明,AP组HepG2细胞核内HIF-1α和胞内VEGF蛋白条带平均灰度值分别是SD组的(9.34±1.67)倍和(3.42±0.83)倍,组间均有统计学差异(P<0.01)。AP组HepG2细胞核内HIF-1DNA结合活性明显高于对照组(P<0.01)。结论:常氧酸性环境能显著提高HepG2细胞HIF-1蛋白含量及其DNA结合活性,同时其下游基因VEGF蛋白含量增加,推测除低氧环境外,酸性环境也可能是导致恶性肿瘤中HIF-1高表达的原因之一。
OBJECTIVE: To observe the effect of acidic environment on the expression and activity of hypoxia-inducible factor (HIF) -1α in hepatoma cells and to explore the role of hypoxia-inducible factor (HIF) -1α in tumor growth. Methods: The HepG2 cells were cultured in vitro. The cell viability was measured by MTT assay after 20 h in normal acidic environment (AP group, pH6.5) and compared with normal cultured cells (SD group, pH7.2) The changes of HIF-1αprotein expression in HepG2cells in AP and SD groups were detected. The changes of HIF-1DNA binding activity in HepG2cell of AP and SD groups were analyzed by transcription factor DNA binding ELISA kit (TransAMTM). Immunocytochemistry and Western blotting was used to detect the protein expression of vascular endothelial growth factor (VEGF) in HepG2 cells in each group. Results: The cell survival rate in AP group was (98.71 ± 1.79)% after treated with normoxic acid (pH6.5) for 20h, which was slightly lower than that in SD group (P> 0.05). Western blot results showed that the average gray value of HIF-1α and VEGF protein bands in HepG2 cells of AP group were (9.34 ± 1.67) times and (3.42 ± 0.83) times of SD group, respectively, with significant difference between the two groups (P <0.01). The HIF-1 DNA binding activity of HepG2 cells in AP group was significantly higher than that in control group (P <0.01). CONCLUSION: Hypoxia environment can significantly increase the HIF-1 protein content and DNA binding activity of HepG2 cells and increase the expression of VEGF protein in HepG2 cells. It is speculated that in addition to hypoxia, the acidic environment may also lead to HIF-1 High expression of one of the reasons.