摘要
目的:探讨人参皂苷Rh2诱导人急性T淋巴细胞白血病(T-cell acute lymphoblastic leukemia,T-ALL)Jurkat细胞的凋亡及其机制。方法:采用四甲基偶氮唑蓝(MTT)法检测不同浓度的Rh2(0、10、20、40和80μg/ml)对Jurkat细胞增殖活性的影响,并计算48h下Rh2对Jurkat细胞的半抑制浓度(IC50);采用形态学方法及Hoechst33258荧光染色观察IC50剂量下Rh2共培养48h的Jurkat细胞凋亡状况。后将细胞实验分为4组:对照组、Rh2(IC50)组、PI3K抑制剂LY294002(50μmol/l)组以及Rh2(IC50)+LY294002(50μmol/l)组。同步培养48h后,采用PI单染和AnnexinV-FITC/PI双染分别检测Jurkat细胞凋亡及细胞周期变化;采用Western blot检测各组Jurkat细胞凋亡相关蛋白BAX、BCL-2、Cleaved-Caspase3,细胞周期相关蛋白CyclinD1以及PI3K/AKT信号通路相关蛋白AKT、p-AKT的表达水平。结果:Rh2(10-80μg/ml)呈剂量-时间依赖性抑制Jurkat细胞增殖(r48h=0.999,P<0.01;r80μg/ml=0.991,P>0.05),并伴有明显的细胞凋亡形态学改变。流式细胞术检测结果显示,与对照组比较,Rh2组和LY294002组细胞凋亡率显著增加,且细胞多数被阻滞在G0/G1期;而与Rh2组和LY294002组比较,Rh2+LY294002组细胞凋亡及细胞阻滞现象更为显著。Western blot结果显示,与对照组比较,Rh2能显著促进BAX、Cleaved-Caspase3蛋白表达,抑制BCL-2、CyclinD1及p-AKT的表达,且LY294002对这一效应具有显著的促进作用。结论:Rh2能够呈时间-剂量依赖性地诱导Jurkat细胞的凋亡;且能对Jurkat细胞产生明显的G0/G1期阻滞;这可能与Rh2抑制PI3K/AKT通路进而介导的一系列凋亡信号级联反应密切相关。
Objective:To investigate the apoptosis-inducing effect of Ginsenoside (Rh2) on human acute T lymphoblastic leukemia Jurkat cells and it mechamism. Methods:The effects of different concentration of Rh2 (0,10, 20,40 and 80 μg/ml) on the proliferation activity of Jurkat cells were detected by methyl thiazolyl tetrazolium (MTT) method,and the semi-inhibitory concentration (IC50) of Rh2 on Jurkat cells at 48 h was calculated. Microscopy and Hoechst 33258 fluorescence staining were used to observe the apoptosis of Jurkat cells treated with IC50 Rh2 for 48 h. And then,the cell experiment was divided into 4 groups:control,Rh2 (IC50),PI3K inhibitor LY294002 (50 μmol/l) and Rh2 (IC50)+ LY294002 (50 μmol/l). After synchronous culture for 48 h,the apoptosis and cycle changes of Jurkat cells were detected by using PI single staining and Annexin V-FITC/PI double staining,respectively. Western blot was used to detect the expression level of apoptosis-related protein BAX,BCL-2,Cleaved-Caspasase 3,cell cycle-related protein Cyclin D1 and PI3K/AKT signaling pathway-related protein AKT and p-AKT. Results:Rh2 (10-80 μg/ml) inhibited the Jurkat cell proliferation in a dose-time dependent manner (r48h = 0.999,P < 0.01;r80 μg/ml = 0.991;P > 0.05),accompanied by obvious morphological changes of apoptosis cells. Flow cytometry showed that compared with the control group,the cell apoptosis rate in Rh2 or LY294002 group significantly increased,and the cell cycle was mostly blocked in G0/G1 phase. However,the cell apoptosis and cell cycle block in Rh2+LY294002 group were more significant than that in Rh2 and LY294002 group. Western blot showed that compared with the control group,Rh2 significantly promoted the expression of BAX and Cleaved-Caspasase 3,inhibited the expression of BCL-2,Cyclin D1 and p-AKT,furthermore LY294002 significantly promoted this effect. Conclusion:Rh2 can induce the apoptosis of Jurkat cells in time-dose dependent manner,moreover,Rh2 also can result in an obvious block of Jurkat cells at G0/G1,that may be closel
作者
聂柳
彭罕鸣
NIE Liu;PENG Han-Ming(Department of Gastroenterology,Children′s Hospital Affiliated to Tongji Medical School,Huazhong University of Science and Technology,Wuhan 430000,Hubei Province,China)
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2019年第4期1111-1117,共7页
Journal of Experimental Hematology
