大黄素抑制HL-60/ADR耐药细胞增殖和诱导凋亡的作用  

Inhibitory Effects of Emodin on Drug-resistant HL-60/ADR Cell Proliferation and Its Induction of Apoptosis

作　　者：陈英玉[1] 郑合勇[1] 胡建达[1] 郑志宏[1] 郑静[1] 连晓岚[1] 吕联煌[1] 

机构地区：[1]福建医科大学附属协和医院福建省血液病研究所,福州350001

出　　处：《中国实验血液学杂志》2007年第5期955-960,共6页Journal of Experimental Hematology

基　　金：福建省科技项目三项费(编号2002Y048);福建省医学创新课题(编号2001-CX-02);福建省高校新世纪优秀人才支持计划(编号NCEFJ-0604);福建医科大学校级课题(编号JS06081)资助

摘　　要：本研究探讨中药大黄素(emodin)对人白血病耐药细胞株HL-60/ADR的增殖、凋亡影响及其相应的可能作用机制。采用MTT法绘制细胞生长曲线;集落培养法观察大黄素对HL-60/ADR克隆形成的影响;细胞周期分析、线粒体跨膜电位检测、Caspase-3酶活性检测、Annexin V FITC/PI法、TdT酶介导的原位缺口末端标记法(TUNEL)分析凋亡细胞;RT-PCR及Western blot法检测大黄素作用后不同时间段bcl-2、c-myc mRNA及Bcl-2、c-Myc、Caspase-3前体蛋白表达水平的变化。结果表明,大黄素对HL-60/ADR细胞增殖具有明显的抑制作用,呈时间和浓度依赖性。较低浓度大黄素即可抑制HL-60/ADR细胞集落形成,IC50值为5.79μmol/L。细胞周期分析显示,与对照组比较,40、80μmol/L浓度组细胞被阻滞于G0/G1期比率明显增高(p<0.01),G2/M期细胞比率降低(p<0.01),而20μmol/L浓度组细胞周期改变差异不具有显著性(p>0.05),各浓度组均检测到典型的亚二倍体峰(凋亡峰)。大黄素作用12小时HL-60/ADR细胞线粒体跨膜电位降低,Annexin V FITC/PI法检测到早期凋亡细胞,作用24小时caspase-3酶活性显著升高,作用48小时TUNEL法检测到晚期凋亡细胞,细胞凋亡率呈药物浓度依赖性。大黄素作用HL-60/ADR细胞不同时间段,bcl-2、c-myc mRNA和Bcl-2、c-Myc、Caspase-3前体蛋白表达水平均有不同程度下调,并呈时间依赖性。结论:大黄素能够有效抑制HL-60/ADR细胞增殖,并诱导其凋亡,bcl-2、c-myc表达水平下调,线粒体跨膜电位水平降低和caspase-3激活可能参与了该过程。The study was aimed to investigate the effects of emodin on the proliferation and apoptosis of adriamycin-resistant HL-60/ADR cells, and to explore the underlying mechanism. The cell viability and colony formation were detected by MTT assay and colony formation assay respectively. Apoptotic cells were tested by means of cell cycle analysis, mitochondrial transmembrane potential levels, caspase-3 activity detection, Annexin V FITC/PI staining and TUNEL labeling. RT-PCR was used to analyze the bcl-2 and c-myc mRNA expressions. The protein expressions of Bcl- 2,c-Myc and caspase-3 precursor were determined by Western blot. The results showed that HL-60/ADR cell growth was significantly inhibited by emodin in dose and time dependent manners. Cell colony formation obviously decreased with IC50 5.79 μmol/L. G0/G1 phase cell population increased while G2/M phase cells decreased in 40 and 80 μmol/L groups compared with control group （p 〈0.01 ）, and no significant difference of cell cycle was observed in 20 μmol/L group（p 〉 0.05）. The typical hypo-diploid peak （apoptotic peak） appeared in each dose group. The levels of mitochondrial transmembrane potential of HL-60/ADR cells decreased and caspase-3 activity increased when incubated with emodin for 12 and 24 hours respectively. Apoptosis occured in a dose-dependent manner,and its earlier and later stages were identified by Annexin-V FITC/PI staining and TUNEL labeling methods respectively. The expressions of bcl-2, c- myc mRNA and Bcl-2, c-Myc, caspase-3 precursor protein were all down-regulated in a time-dependent manner after treatment with emodin at different times. It is concluded that emodin efficiently inhibits growth and induces apoptosis on HL-60/ADR cells, which may be related with the down-regulation of mitochondrial transmembrane potential and expressions of bcl-2 and c-myc, as well as up-regulation of caspase-3 activity.

关 键 词：大黄素 HL-60/ADR细胞 细胞增殖 细胞凋亡 

分 类 号：R979.1[医药卫生—药品] R329.8[医药卫生—药学]