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目的探讨HIF-1α表达在体外对肝癌细胞凋亡的影响。方法利用Tet-on基因表达系统调控HePG2细胞HIF-1α的表达,观察细胞凋亡。结果酶切和DNA测序证实Tet-on基因表达系统反应质粒PTRE-HIF-1α构建成功。获得了受强力霉素调控、稳定表达HIF-1α的肝癌细胞。经阿霉素诱导凋亡后,强力霉素终浓度为0μg／ml、0．02μg／ml、0．2μg／ml、1μg／ml、2μg／ml、5μg／ml时的细胞凋亡率分别为59．6％、50．9％、38．1％、30．5％、23．9％、18．3％;强力霉素0．02μg／ml、0．2μg／ml、1μg／ml、2μg／ml、5μg／ml处理细胞后,Casepase-3活性分别降低了2．3、5．5、8．7、12．6、18．8倍。RT- PCR检测结果显示,随着强力霉素浓度的增加,HIF-1α表达水平增加,Survivin和Bcl-2基因表达强度逐渐增加,差异有统计学意义(P<0．001)。结论HIF-1α基因在体外可以抑制肝癌细胞凋亡,而且随着HIF-1α表达水平的增加,对肝癌细胞的凋亡抑制作用进一步加强,可能与HIF-1α诱导survivin和Bcl-2的表达及抑制casepase-3的活性有关。
Objective To investigate the effect of HIF-1α expression on the apoptosis of hepatoma cells in vitro. Methods Tet-on gene expression system was used to regulate the expression of HIF-1α in HepG2 cells and apoptosis was observed. Results The restriction enzyme digestion and DNA sequencing confirmed the successful construction of the Tet-on gene expression system response plasmid PTRE-HIF-1α. Hepatoma cells stably expressing HIF-1α under the control of doxycycline were obtained. After apoptosis induced by doxorubicin, the apoptosis rates of doxycycline were 0 μg/ml, 0.02 μg/ml, 0.2 μg/ml, 1 μg/ml, 2 μg/ml, and 5 μg/ml respectively. 59.6%, 50.9%, 38.1%, 30.5%, 23.9%, 18.3%; Doxycycline 0.02 μg/ml, 0.2 μg/ml, 1 μg/ml, 2 μg After treatment with 5 μg/ml cells, the activity of Casepase-3 was decreased by 2.3, 5.5, 8.7, 12.6, and 18.8 times, respectively. The results of RT-PCR showed that as the doxycycline concentration increased, the expression of HIF-1α increased, and the expression of Survivin and Bcl-2 gene increased gradually. The difference was statistically significant (P<0.001). Conclusion HIF-1α gene can inhibit the apoptosis of hepatoma cells in vitro. With the increase of the expression of HIF-1α, the inhibitory effect of HIF-1α on hepatoma cells is further enhanced, which may be related to the expression of survivin and Bcl-2 induced by HIF-1α. Inhibition of casepase-3 activity is relevant.