IWP －2对人肝癌 Hep3B 细胞增殖和凋亡的影响及其机制探讨

摘要

Objective:To study the effects of Wnt/β-catenin inhibitor IWP -2 on the proliferation and apoptosis of human hepatic carcinoma cell lines Hep3B,and to elucidate the possible mechanism.Methods:Human hepatic car-cinoma Hep3B cells were treated with IWP -2 (0,20,40,80,160,320μmol/L).The inhibitory effect of IWP -2 on cell proliferation was examined by CCK -8.The cell cycle distribution and apoptosis rate of Hep3B cells induced by IWP -2 were detected by flow cytometry(FCM).The expression of apoptotic -related protein was detected by West-ern blotting.The expression levels of Cyclin D1 and survivin proteins were detected by Western blotting.Cyclin D1 and survivin mRNA expression were determined by Real time polymerase chain reaction(RT -PCR).Results:The proliferation of cells was inhibited in a concentration -and time -dependent manner when it was treated with IWP -2(20 ~320μmol/L).Absorbance values between groups had statistically significant difference(P <0.01).FCM re-sults showed that the proportion of cells in G0 /G1 phase were increased while which in S phase were decreased by IWP -2(20 ~320μmol/L)for 48h.The apoptosis rate of Hep3B cells induced by IWP -2 for 48h was incresed in a concentration -dependent manner.Absorbance values between groups had statistically significant difference(P <0.01).The expression levels of caspase -3,caspase -7 proteins could be increased by IWP -2 in a concentration -manner.The expression levels of Cyclin D1 and survivin proteins and mRNA were down -regulated by IWP -2 in a concentration -manner.Conclusion:IWP -2 can inhibited the proliferation of Hep3B cells and induces apoptosis of Hep3B cells.These effects may related with the down -regulation of Cyclin D1 and survivin.%目的：研究 Wnt／β－catenin 抑制剂 IWP －2对体外培养的人肝癌细胞 Hep3B 细胞增殖和凋亡的影响，并探讨其可能的作用机制。方法：用终浓度分别为0、20、40、80、160、320μmol／L 的 IWP －2作用于人肝癌Hep3B 细胞，采用 CCK －8法检测细胞的增殖抑制率，FCM法检测细胞周期分布与细胞凋亡率，Western blot法检测细胞凋亡蛋白的表达，Western blot 法检测细胞中 Cyclin D1和 survivin 蛋白表达的变化，实时定量 RT－PCR 法检测 Cyclin D1和 survivin mRNA 表达的变化。结果：20～320μmol／L 的 IWP －2对细胞的增殖均有抑制作用，其抑制作用随 IWP －2浓度的升高和作用时间的增长而增强，各组与对照组相比差异有统计学意义（P ＜0．01）。FCM检测结果显示，IWP －2（20～320μmol／L）能够导致 G0／G1期细胞比例上升，S 期细胞比例下降；同时诱导细胞凋亡，凋亡率随 IWP －2浓度的升高而上升，与对照组相比差异有统计学意义（P＜0．01）。IWP －2可引起 caspase －3和 caspase －7蛋白表达水平的升高，并且可下调肝癌细胞中 Cyclin D1和 survivin 基因和蛋白的表达水平，呈明显的剂量依赖性。结论：IWP －2可抑制 Hep3B 细胞的增殖并诱导其凋亡，其机制可能与抑制 Cyclin D1和 survivin 的表达有关。