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背景:已有研究证明,冬凌草乙素可以通过诱导许多实体肿瘤的细胞凋亡而发挥抗肿瘤作用,目前,冬凌草乙素对白血病HL-60细胞的作用尚未见资料报道。目的:观察冬凌草乙素对白血病HL-60细胞的体外增殖抑制作用及其机制。方法:以不同浓度的冬凌草乙素(10~50μmol/L)作用于体外培养的HL-60细胞24,48,72h,应用MTT法检测细胞生长抑制率,流式细胞术检测细胞周期,免疫印迹法检测Caspase-3及其裂解底物多聚(ADP-核糖)聚合酶(PARP)表达水平的变化,并对细胞周期调节蛋白P21、P16的表达水平进行检测。结果与结论:冬凌草乙素可显著抑制细胞的生长及诱导细胞发生凋亡,呈现出明显的量-效与时-效关系。流式细胞术检测结果表明细胞主要被阻滞在G0/G1期,50μmol/L的冬凌草乙素作用48h后可以出现典型的亚G1期峰(细胞凋亡峰)。免疫印迹法检测结果显示Mr32000的Caspse-3酶被活化出现Mr20000亚单位片段,同时Caspse-3的底物PARP被裂解出现Mr89000的亚单位片段,免疫印迹法检测结果还显示50μmol/L的冬凌草乙素作用不同时间后,细胞周期调节蛋白P21及P16的表达水平逐渐升高。结果提示冬凌草乙素在体外对HL-60细胞具有显著的细胞周期阻滞作用,并诱导细胞发生凋亡,通过上调细胞周期调节蛋白P21及P16的表达水平及激活Caspse-3可能是冬凌草乙素引起HL-60细胞G0/G1阻滞及诱导细胞发生凋亡的重要作用机制之一。
BACKGROUND: Studies have shown that R. oryzepin can exert its anti-tumor effects by inducing apoptosis of many solid tumors. At present, the effect of R. oryzepin on leukemia HL-60 cells has not been reported. OBJECTIVE: To observe the inhibitory effect of Oridonin on leukemia HL-60 cells in vitro and its mechanism. Methods: The HL-60 cells cultured in vitro were treated with different concentrations of Oridonin (10~50μmol/L) for 24, 48 and 72 hours. The cell growth inhibition rate was detected by MTT assay, and the cell cycle was detected by flow cytometry. The expression of Caspase-3 and its cleavage substrate poly(ADP-ribose) polymerase (PARP) was detected by Western blot, and the expression levels of P21 and P16 were detected. RESULTS AND CONCLUSION: Oridonin can significantly inhibit cell growth and induce apoptosis of cells, showing a significant dose-effect and time-effect relationship. The results of flow cytometry showed that the cells were mainly arrested in G0/G1 phase, and typical sub-G1 phase peaks (apoptosis peaks) could appear after treatment with 50 μmol/L oridonin for 48 h. The results of immunoblotting showed that the Mr20000 subunit fragment was activated by the activation of the Caspase-3 enzyme in Mr32000, and the subunit fragment of Mr89000 was cleaved by the substrate PARP of Caspse-3. The immunoblot test results also showed that 50 μmol/L of the winter ling The effect of thiophosin on the expression of P21 and P16 increased gradually. The results suggest that oridonin has a significant cell cycle arrest effect on HL-60 cells in vitro and induces cell apoptosis. Up-regulation of the expression of the cell cycle regulatory proteins P21 and P16 and activation of Caspse-3 may be the winter Pryazoidin induces G0/G1 arrest and induces apoptosis in HL-60 cells.