摘要
目的:观察青篙琥酯(ART)对骨髓增生异常综合征(MDS)细胞系SKM-1细胞增殖的影响,并探讨其相关作用机制。方法:不同浓度ART处理SKM-1细胞后,应用CCK-8实验检测细胞活力;流式细胞术测定细胞凋亡及细胞周期分布;DCFH-DA荧光探针检测细胞内活性氧簇的变化,Fluo-3-Am荧光探针检测细胞内的钙离子浓度的变化;Western blot方法检测凋亡相关蛋白BCL-2、BAX、BAD、P-BAD、survivin和XIAP的表达水平变化。结果:ART能明显抑制SKM-1细胞增殖,其效应呈剂量和时间依赖性(r=-0.841;r=-0.786);抗氧化剂Trolox预处理能显著减轻ART对SKM-1细胞的抑制;caspase抑制剂Ac-REVD-CHO能部分减少ART对SKM-1细胞的抑制。ART处理SKM-1细胞24 h后,可诱导SKM-1细胞凋亡,并使细胞周期阻滞于G_0/G_1期;ART可诱导SKM-1细胞内钙离子和活性氧簇水平明显升高。Western blot结果显示,ART显著下调SKM-1细胞P-BAD和Survivin的蛋白表达水平,其下降程度与ART剂量呈高度负相关(r=-0.909;r=-0.849);相反,ART对SKM-1细胞BAD和XIAP蛋白表达水平无明显影响;尽管BCL-2蛋白表达在ART处理组与对照组相比无显著差别,但BCL-2/BAX比值与对照组相比显著下降,其下降程度与ART剂量呈高度负相关(r=-0.866)。结论:ART能明显抑制SKM-1细胞增殖和诱导细胞凋亡,使细胞周期阻滞于G_0/G_1期。ART抗MDS作用机制与其提高细胞内钙离子浓度和活性氧簇水平有关。此外,ART促SKM-1细胞凋亡作用主要与BCL-2/BAX比值下降、BAD磷酸化水平减低和survivin表达水平下调有关。
Objective:To investigate the effects of artesunate(ART) on proliferation,cell cycle and apoptosis of SKM-L cells in vitro and to explore the underlying mechanisms.Methods:After SKM-L cells were treated with different concentrations of ART,the cell proliferation was determined by CCK-8 method.Apoptosis and distribution of cell cycle were detected by flow cytometry.Both DCFH-DA fluorescent probe and Fluo-3-Am fluorescent probe were used to detect the changes of intracellular reactive oxygen species(ROS) and calcium ion concentration.Western blot was used to measure the protein levels of BCL-2,BAX,BAD,P-BAD,survivin and XIAP.Results:ART obviously inhibited the growth of SKM-L cells in time and dose-dependent manners(r =- 0.841;r = 0.- 786).The antioxidant troloxpretreatment significantly decreased the growth inhibition effect of ART on SKM-L cells.Caspase inhibitor Ac-DEVDCHO partially reduced the growth inhibition effect of ART on SKM-L cells.After treatment with ART for 24 hours,the apoptosis of SKM-L cells was found,the cell cycle of SKM-L was arrested in G_0/G_1 phase,ART could elevate the levels of calciumion and reactive orygen.ART could significantly down-regulate the protein expression levels of P-BAD and survivin in SKM-L cells,and showed a highly negative correlation with ART dose(r =-0.909;r =-0.849).On the contrary,ART had no significant effect on expression levels of BAD and XIAP in SKM-L cells,and after ART treatment,although BCL-2 protein expression was not significantly different when compared with control group,but the BCL-2/BAX ratio significantly decreased and highly negatively correlated with ART dose(r =-0.866).Conclusion:The ART significantly suppresses the cell proliferation,induces the apoptosis and promoted cell cycle arrest at G_0/G_1phase in SKM-L cells.The mechanisms of ART anti-MDS is associated with the increase of intracellular calciumion concentration and ROS levels.In addition,the pro-apoptotic activity of ART may be involved in the regulation of BCL-2 /BAX ratio and the expressions of P-bad and survivin.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2016年第1期131-137,共7页
Journal of Experimental Hematology
基金
河北省医学科学研究课题计划立项(20130186)
