CAJ | 학술논문

目的研究DNA引物酶抑制剂碘化-3,3'-二乙基-9-甲基-硫杂羰花青(DMTCCI)诱导人粒细胞性白血病HL-60细胞凋亡并探索其机制.方法分别采用不同浓度的DMTCCI处理培养于RPMI-1640培养基的HL-60细胞.采用MTT法检测DMTCCI对HL-60细胞的生长抑制作用.采用流式细胞仪和DNA琼脂糖凝胶电泳方法检测细胞凋亡.采用蛋白免疫印迹(Western blotting)法观察捌亡相关蛋白survivin,Bcl-xL,Bad,Bax,Bcl-2,caspase-9,caspase-3,caspase-6,PARP,DFF45和lamin B的表达.采用ApoAlert Caspase-3分析试剂盒检测caspase-3的活性.结果 DMTCCI具有抑制人白血病HL-60细胞增殖的作用,其IC50值为0.24μmol·L-1.流式细胞仪和DNA琼脂糖凝胶电泳结果显示,DMTCCI可诱导HL-60细胞凋亡.在经DMTCCI处理的HL-60细胞中,survivin和Bcl-xL蛋白的表达水平下调,Bad和Bax蛋白的表达水平上调,Bcl-2蛋白的表达水平无变化,caspase-9,caspase-3,caspase-6,PARP, DFF45和lamin B被分别裂解,产生相应裂解产物.在HL-60细胞中,caspase-3的活性在1μmol·L-1 DMTCCI处理3 h时明显升高,在处理12 h时达到最高峰.结论 DMTCCI可抑制人白血病HL-60细胞的增殖并诱导其发生细胞凋亡.Bcl-2家族蛋白、survivin和caspases家族蛋白可能参与了上述诱导HL-60细胞凋亡的过程.
목적 연구DNA인물매억제제전화-3,3'-이을기-9-갑기-류잡탄화청(DMTCCI)유도인립세포성백혈병HL-60세포조망병탐색기궤제.방법 분별채용불동농도적DMTCCI처리배양우RPMI-1640배양기적HL-60세포.채용MTT법검측DMTCCI대HL-60세포적생장억제작용.채용류식세포의화DNA경지당응효전영방법검측세포조망.채용단백면역인적(Western blotting)법관찰팔망상관단백survivin,Bcl-xL,Bad,Bax,Bcl-2,caspase-9,caspase-3,caspase-6,PARP,DFF45화lamin B적표체.채용ApoAlert Caspase-3분석시제합검측caspase-3적활성.결과 DMTCCI구유억제인백혈병HL-60세포증식적작용,기IC50치위0.24μmol·L-1.류식세포의화DNA경지당응효전영결과현시,DMTCCI가유도HL-60세포조망.재경DMTCCI처리적HL-60세포중,survivin화Bcl-xL단백적표체수평하조,Bad화Bax단백적표체수평상조,Bcl-2단백적표체수평무변화,caspase-9,caspase-3,caspase-6,PARP, DFF45화lamin B피분별렬해,산생상응렬해산물.재HL-60세포중,caspase-3적활성재1μmol·L-1 DMTCCI처리3 h시명현승고,재처리12 h시체도최고봉.결론 DMTCCI가억제인백혈병HL-60세포적증식병유도기발생세포조망.Bcl-2가족단백、survivin화caspases가족단백가능삼여료상술유도HL-60세포조망적과정.
Aim To investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide(DMTCCI) , an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells. Methods HL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition.Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit. Results DMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0. 24 μmol · L-1. The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated,while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 μmol · L-1 of DMTCCI in HL-60 cells. Conclusion DMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might playa role in the apoptosis process induced by DMTCCI.