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【目的】探讨吴茱萸碱(EVO)联合细胞周期蛋白依赖性激酶1(CDK1)的特异抑制剂RO3306对人肝癌细胞HepG2的生长抑制、诱导凋亡是否具有协同增效作用。【方法】采用四甲基偶氮唑盐(MTT)法、碘化丙啶(PI)单染流式细胞术检测EVO诱导不可逆凋亡的时间转折点;采用MTT法、集落形成法、PI单染流式细胞术检测EVO和RO3306联合用药与单独用药比较是否具有协同增效作用(q>1.15为协同作用)。【结果】MTT法结果显示:作用16 h起,再撤药培养12 h与撤药前比较抑制率明显上升。流式细胞术检测表明:细胞同步化处理后吴茱萸碱作用12 h大部分细胞发生M期阻滞(M-arrest);作用20 h细胞发生M期滑移(M-slippage)。MTT法检测2μmol/L吴茱萸碱、2μmol/L RO3306及两者联合作用的抑制率分别是33.64%、6.10%和52.74%,q值为1.32。集落形成法显示:1μmol/L吴茱萸碱、2μmol/LRO3306和两者联合作用的抑制率分别是48.21%、11.13%和78.13%,q值为1.83;2μmol/L吴茱萸碱、2μmol/LRO3306和两者联合作用的抑制率分别是9.75%、66.8%和91.2%,q值为1.3。流式细胞术显示:2μmol/L吴茱萸碱、2μmol/L RO3306和两者联合作用的抑制率分别是23.7%、6.6%和44.8%,q值为1.31。【结论】吴茱萸碱诱导M-arrest发生于12 h左右,诱导不可逆凋亡的时间转折点为20 h左右,吴茱萸碱联合RO3306对HepG2具有明显的协同抑制作用。
【Aim】 To investigate whether synergistic effect of evodiamine combined with specific inhibitor of cyclin dependent kinase 1 (CDK1) on human hepatocellular carcinoma cell line HepG2 was induced by RO3306. 【Methods】 MTT assay and propidium iodide (PI) single flow cytometry were used to detect the time-point of EVO-induced irreversible apoptosis. MTT assay, colony formation assay, PI single staining Flow cytometry was used to determine whether synergistic effects of EVO and RO3306 in combination with monotherapy were obtained (q> 1.15 synergistic). 【Results】 The results of MTT assay showed that the inhibition rate increased significantly after 16 h of treatment and 12 h after withdrawal and withdrawal. Flow cytometry showed that most of the cells evolved M-arrest after evodiamine treatment for 12 h, and M-slippage occurred at 20 h. The inhibitory rates of 2μmol / L evodiamine, 2μmol / L RO3306 and the combination of the two were 33.64%, 6.10% and 52.74% respectively with MTT assay and q value was 1.32. The colony formation assay showed that the inhibitory rates of 1μmol / L evodiamine, 2μmol / LRO3306 and the combination of the two were 48.21%, 11.13% and 78.13%, respectively, and q values were 1.83, 2μmol / L evodiamine, 2μmol / LRO3306 and The inhibitory rates of combined action were 9.75%, 66.8% and 91.2%, respectively, and the q value was 1.3. Flow cytometry showed that the inhibitory rates of 2μmol / L evodiamine, 2μmol / L RO3306 and their combination were 23.7%, 6.6% and 44.8% respectively, and the q value was 1.31. 【Conclusion】 Evodiamine-induced M-arrest occurred at about 12 h, and the time turning point for inducing irreversible apoptosis was about 20 h. Evodiamine combined with RO3306 had a significant synergistic inhibitory effect on HepG2.