苦参碱联合LY294002对人髓系白血病K562细胞增殖、凋亡和细胞周期的影响  

Effects of matrine combined with LY294002 on proliferation,apoptosis and cell cycle of human myeloid leukemia K562 cells

作　　者：郝艳梅[1] 纪俊莉 刘纯艺 张楠[1] 宫雅娟 HAO Yanmei;JI Junli;LIU Chunyi;ZHANG Nan;GONG Yajuan(Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis,Bengbu Medical College,Bengbu 233030,China)

机构地区：[1]蚌埠医学院肿瘤基础研究与临床检验诊断重点实验室,安徽蚌埠233030

出　　处：《南方医科大学学报》2022年第11期1739-1746,共8页Journal of Southern Medical University

基　　金：国家大学生创新训练项目(202010367049);安徽省教育厅重点科学项目(KJ2021A0691)。

摘　　要：目的探究苦参碱(Mat)联合LY294002对人髓系白血病K562细胞增殖、凋亡和细胞周期的影响及可能机制。方法采用CCK-8法检测不同浓度Mat单药和联合LY294002对K562细胞增殖的影响;将K562细胞分为对照组、Mat组、LY294002组和Mat+LY294002组,分别采用0.4 g/L Mat及10μmol/L LY294002单独或联合干预48 h后,应用光学显微镜观察细胞形态变化,流式细胞术annexinⅤ-FITC/PI双标记检测细胞凋亡,PI单标记检测细胞周期变化,Western blot法检测Mat联合LY294002对K562细胞p-mTOR、p-PI3K、Akt、p-Akt、CyclinD1、Bcl-2、Caspase-9蛋白表达的影响。结果CCK-8结果显示,不同浓度Mat单药和联合LY294002均可抑制K562细胞的增殖,具有时间和浓度依赖性,且联合组增殖抑制率比单药组明显升高(P<0.01)。形态学检查显示,联合用药组比单药组凋亡表现更明显。流式细胞结果显示,与对照组及单药组相比,联合组显著促进细胞凋亡[(42.50±2.63)%,P<0.01],显著增加了细胞周期G0/G1期细胞比率[(57.23±1.44)%,P<0.01]。Western blot结果表明,两药联合后K562细胞p-mTOR、CyclinD1、p-PI3K、p-Akt、Bcl-2蛋白表达水平明显增高(P<0.01),Caspase-9蛋白表达水平下降(P<0.01)。结论Mat和LY294002联合应用可以增强对K562细胞生长抑制的协同作用,其机制可能是通过有效下调PI3K/Akt信号通路中的p-Akt表达,使p-mTOR、CyclinD1和Bcl-2蛋白表达下调,Caspase-9蛋白表达上调来实现的。Objective To investigate the effects of matrine combined with LY294002 on proliferation,apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism.Methods The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay.The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10μmol/L Y294002,either alone or in combination,and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling;the changes in cell cycle was detected with PI labeling.Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR,p-PI3K,Akt,p-Akt,cyclinD1,Bcl-2 and caspase-9 in the cells.Results Treatment with different concentrations of matrine,both alone and in combination with LY294002,inhibited the proliferation of K562 cells in a time-and concentration-dependent manner.Compared with matrine treatment alone,the combined treatment caused more obvious morphological changes of the cells,significantly increased cell apoptosis(P<0.01),and induced cell cycle arrest in G0/G1(P<0.01).Western blotting showed that the protein expression levels of p-mTOR,cyclinD1,p-PI3K,p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment(P<0.01).Conclusion Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway,reducing the expressions of p-mTOR,cyclinD1 and Bcl-2,and increasing the expression of caspase-9.

关 键 词：急性髓系白血病 苦参碱 LY294002 细胞凋亡 细胞周期 AKT 

分 类 号：R285[医药卫生—中药学]