蟾蜍灵诱导HL-60细胞凋亡过程中对Bcl-2、Survivin、Smac/DIABLO表达的影响  

Downregulation of Bcl-2 and survivin expression and release of Smac/DIABLO involved in bufalin-in-duced HL-60 cell apoptosis

作　　者：田昕[1] 罗颖[1] 刘云鹏[1] 侯科佐[1] 金波[1] 张敬东[1] 王甦[1] 

机构地区：[1]中国医科大学附属第一医院肿瘤内科,沈阳110001

出　　处：《中华血液学杂志》2006年第1期21-24,共4页Chinese Journal of Hematology

基　　金：辽宁省自然科学基金资助项目(20022077)辽宁省教育厅高等学校科学研究基金资助项目(20122152)

摘　　要：目的研究蟾蜍灵在诱导HL-60细胞凋亡过程中,对线粒体凋亡途径相关基因Bcl-2、 Bax、Survivin及Smac/DIABLO表达的影响。方法采用锥虫蓝拒染法检测细胞存活情况,细胞形态学观察及流式细胞术检测细胞凋亡,Western blot分析Bcl-2、Bax、Smac/DIABLO、Survivin及caspase-3蛋白表达,RT-PCR检测Survivin mRNA表达。结果蟾蜍灵抑制HL-60细胞增殖,24,48及72 h的IC50 值分别为25．8,8．0及2．1 nmoL/L。蟾蜍灵大于50．0 nmol/L时可明显诱导HL-60细胞凋亡。50．0 nmol/L蟾蜍灵作用6,12,24及48 h,Bcl-2蛋白表达分别下调至作用前的88．6％,53．3％,19．2％及 9．5％,而Bax蛋白表达无明显变化,Bcl-2/Bax比值由2．0分别降至1．7,1．1,0．4及0．2。Survivin蛋白表达分别下调至作用前的75．2％,54．8％,37．5％及20．3％;Survivin mRNA表达在作用6,12和24 h 后分别下调至作用前的85．7％,39．4％和12．5％,在作用48 h后几乎检测不到。50．0 nmol/L蟾蜍灵作用12,24及48 h,线粒体部分的Smac/DIABLO蛋白表达分别下调至作用前的77．5％,21．2％及 15．3％,而胞浆部分的Smac/DIABLO蛋白表达分别上调至作用前的1．4,2．0．及3．5倍。蟾蜍灵作用 8-48 h可检测到caspase-3的活化亚基。结论蟾蜍灵诱导的HL-60细胞凋亡与Bcl-2及Survivin表达下调、线粒体释放Smac/DIABLO及caspase-3活化有关。Objective To investigate the expression of mitochondria-mediated apoptosis pathway related genes bcl-2, Box, survivin and Smac/DIABLO in bufalin induced HL-60 cell apoptosis. Methods Cell viability was determined by trypan blue exclusion, apoptosis by morphology and flow cytometry, expressions of Bcl-2, Bax, Survivin, Smac/DIABLO and caspase-3 protein by Western blot,and expression of survivin mRNA by RT-PCR. Results Proliferation of HL-60 cells was inhibited by bufalin and the IC50 at 24, 48 and 72 h were 25.8, 8.0 and 2.1 nmol/L, respectively. Apoptosis was induced when the cells were treated with bufalin at concentrations of 50.0 nmoL/L and higher. Compared with the control, HL-60 cells treated with bufalin at 50.0 nmoL/L for 6, 12, 24 and 48 h showed decrease of Bcl-2 protein expression to 88.6%, 53.3%, 19.2% and 9.5%, Bcl-2/Bax ratio （control 2.0） to 1.7, 1.1,0.4 and 0.2, Survivin protein expression to 75.2%, 54. 8%, 37.5% and 20.3%, and survivin mRNA to 85.7%, 39. 4%, 12.5% and 0%, respectively. The expression of Smac/DIABLO protein was downregulated to 77.5% （ 12 h） ,21.2% （24 h） and 15.3% （48 h） in mitochondrial fraction and upregulated to 1.4 - （12h）, 2.0 - （24 h） and 3.5 - folds （48 h） in eytosolic fraction, respectively. The active subunits of caspase-3 were displayed after treatment for 8 h till 48 h. Conclusion Apoptosis induced by bufalin is related to downregulation of expressions of bcl-2 and survivin, decrease of Bcl-2/Bax ratio, mitochondrial release of Smac/DIABLO, and activation of caspase-3. The mitochondria-mediated apoptotic pathway may be involved in the apoptosis induced by bufalin in HL-60 cells.

关 键 词：蟾蜍灵 基因 BCL-2 基因 surivivin 基因 Smac/DIABLO 细胞系 HL-60 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤]