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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(DMTCC I)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DMTCC I处理培养于RPM I-1640培养基的HL-60细胞。采用MTT法检测DMTCC I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(W estern b lotting)法观察凋亡相关蛋白survivin,Bc l-xL,Bad,Bax,Bc l-2,caspase-9,caspase-3,caspase-6,PARP,DFF45和lam in B的表达。采用ApoA lert Caspase-3分析试剂盒检测caspase-3的活性。结果DMTCC I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,DMTCC I可诱导HL-60细胞凋亡。在经DMTCC I处理的HL-60细胞中,survivin和Bc l-xL蛋白的表达水平下调,Bad和Bax蛋白的表达水平上调,Bc l-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-1DMTCCI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。
OBJECTIVE: To study the apoptosis of human leukemia cell line HL-60 induced by DNAse inhibitor 3, 3’-diethyl-9-methyl-thiocarbocyanine (DMTCC I) and explore its mechanism. Methods HL-60 cells cultured in RPM I-1640 medium were treated with different concentrations of DMTCC I. The inhibitory effect of DMTCC I on HL-60 cells was detected by MTT assay. Apoptosis was detected by flow cytometry and DNA agarose gel electrophoresis. The expressions of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lam in were detected by Western blotting B expression. Caspase-3 activity was measured using the ApoA lert Caspase-3 assay kit. Results DMTCC I inhibited the proliferation of human leukemia HL-60 cells with IC50 of 0.24 μmol·L-1. Flow cytometry and DNA agarose gel electrophoresis showed that DMTCC I could induce HL-60 cell apoptosis. In HL-60 cells treated with DMTCC I, the expression of survivin and Bcl-xL protein was down-regulated, the expression of Bad and Bax protein was up-regulated, the expression of Bcl-2 protein was unchanged, the expression of caspase-9 and caspase- 3, caspase-6, PARP, DFF45 and lamin B were cleaved, resulting in the corresponding cleavage products. In HL-60 cells, the activity of caspase-3 was significantly increased at 1 h after treatment with 1 μmol.L-1 DMTCCI and reached the peak at 12 h. Conclusion DMTCCI can inhibit the proliferation of human leukemia HL-60 cells and induce its apoptosis. Bcl-2 family proteins, survivin and caspases family of proteins may be involved in the above process of inducing HL-60 cell apoptosis.