吉西他滨对人肝癌HepG2细胞的抑制作用及其可能机制

摘要

目的 探讨吉西他滨对人肝癌HepG2细胞的抑制作用以及机制. 方法 采用CCK-8法检测细胞的存活率,将不同浓度(0.4,0.8,1.6,3.2μg/mnl)的吉西他滨作用于人肝癌HepG2细胞24 h、48 h,观察细胞的生长抑制作用,根据结果计算得知细胞的半数抑制浓度(IC50)为1.57 μg/ml,然后在1.57 μg/ml的吉西他滨的基础上加入自噬抑制剂3-MA,观察细胞的生长抑制情况,流式细胞术检测吉西他滨作用于人肝癌HepG2细胞后细胞的凋亡情况,荧光显微镜观察MDC染色后细胞自噬的形态学变化,RT-PCR检测自噬相关基因Beclin-1和LC-3表达的变化. 结果 CCK-8结果显示不同浓度的吉西他滨对人肝癌HepG2细胞有明显的抑制作用,呈现时间和浓度的剂量依赖性,用药后细胞存活率明显下降,与对照组相比较差异具有统计学意义(P＜0.01).在1.57μg/mnl吉西他滨作用基础上加入自噬抑制剂后,细胞的存活率明显上升,由(48.7±1.23)％变为(61.22±2.17)％(P＜0.01).流式细胞术结果显示吉西他滨诱导肝癌HepG2细胞发生凋亡,随着吉西他滨浓度升高,细胞的凋亡率明显上升,MDC染色以及实时荧光定量PCR结果显示吉西他滨可以诱导HepG2细胞发生自噬,引起自噬相关基因基因的Beclin-1和LC-3表达的增加. 结论 吉西他滨对人肝癌HepG2细胞有明显的抑制作用,其机制可能通过诱导细胞发生凋亡和自噬来导致细胞最终死亡.%Objective To investigate the inhibitory effect of gemcitabine on human hepatocellular carcinoma HepG2 cells and its possi-ble mechanism.Methods CCK-8 method was used to detect the HepG2 cell growth after treated with different concentrations of gemncitabine(0.4,0.8,1.6,3.2 μg/ml) for 24 h and 48 h.Based on the treatment of gemcitabine(the half inhibitory concentration of the cell,1.57 μg/ml),the autophagy inhibitor 3-MA was added to observe the inhibition of cell growth.The apoptosis of HepG2 cells was detected by flow cytometry after exposed to gemcitabine.The morphological changes of MDC cell autophagy after staining were observed under fluorescence microscopy.RT-PCR was used to detect the expression of autophagy-related genes LC-3 and Beclin-1.Results The results of CCK-8 showed that gemcitabine had a great growth inhibitory effect on hmnan hepatoma HepG2 cells in a concentration-and time-dependent manner.After the administration,the cell survival rate decreased significantly compared with control group (P ＜ 0.01) When adding autophagy inhibitor 3-MA based on 1.57 μg/ml gemcitabine treatment,the survival rate of the cells was significantly increased from (48.7 + 1.23) ％ to (61.22 + 2.17) ％ (P ＜ 0.01).Flow cytometry showed that the apoptosis of hepatoma HepG2 cells increaced after exposed to gemcitabine in a concentration-dependent manner.MDC staining and real-time fluorescence quantitative PCR results showed that gemcitabine induced the autophagy of HepG2 cells and increased the expression of autophagy-related genes Beclin-1 and LC-3.Conclusion Gemcitabine has a significant inhibitory effect on human hepatocellular carcinoma cell line HepG2,which may lead to the eventual death of cells by inducing the apoptosis and autophagy.