四嗪二甲酰胺对白血病细胞株HL-60体外作用的研究  

Effects of N,N'-di-(m-methylphenyi)-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide on HL-60 leukemia cells in vitro

作　　者：周永列[1] 吕亚萍[2] 胡惟孝[2] 邱莲女[1] 王文松[1] 杨忠愚[2] 刘建栋[1] 

机构地区：[1]浙江省人民医院中心实验室 [2]浙江工业大学药学院

出　　处：《中国临床药理学与治疗学》2006年第9期1006-1012,共7页Chinese Journal of Clinical Pharmacology and Therapeutics

基　　金：浙江省科技厅资助项目(№2003c33019)

摘　　要：目的：观察四嗪二甲酰胺（ZGDHu-1）体外抑制白血病细胞株HL-60增殖，诱导细胞分化和凋亡作用，并对其作用机制进行初步探讨。方法：将不同浓度的ZGDHu-1与HL-60细胞在体外培养，台盼蓝染色、MTT比色法观察对HL-60细胞增殖的抑制作用；用细胞形态学、DNA凝胶电泳、DNA含量及细胞周期分析、Annexin-V／PI双标记和DNA片段原位末端标记法等分析细胞凋亡。通过NBT试验、细胞表面抗原CD11b、CD13、CD14、CD64和细胞形态学检测ZGDHu-1对HL-60细胞的分化作用。用Rh123／PI测定线粒体跨膜电位（△ψm），用流式细胞术检测Bcl-2、Box、P53、Fas和线粒体膜蛋白的表达变化。结果：ZGDHu-1呈现作用时间和剂量的量效性抑制HL-60细胞增殖和活力，10ng·ml^-1 ZGDHu-1作用HL-60细胞24～72h后，MTr法检测细胞增殖抑制率分别为（7．3±0．3）％、（15．8±1．0）％和（21．3±1．2）％，均显著高于对照组（P〈0．01）；48h、72h的IC50分别为180、180ng·ml^-1。HL-60细胞经ZGDHu-1作用后，大部分细胞阻滞于G2-M期，G0/1期细胞减少；出现典型的细胞形态改变，DNA片端化，经50～1000ng·ml^-1的ZGDHu-1作用后，SubG1由（3.2±1．6）％上升至（67．7±5.9）％，与对照组（0．7±0.5）％比较，具有显著性差异（P〈0．05），Annexin-V／PI标记升高，TUNEL染色后的凋亡细胞的特征性改变等均证实ZGDHu-1能诱导HL-60细胞凋亡。HL-60细胞经10～100ng·ml^-1 ZGDHu-1作用3d后，细胞发生部分分化，NBT阳性率增加，细胞表面CD11b、CD13、CD14、CD64表达增加。ZGDHu-1诱导HL-60细胞凋亡和分化过程中，Bcl-2表达无变化，但Box表达显著增加，Bax／Bcl-2的比值升高，Fas表达增强，而P53表达无变化。随ZGDHu-1作用的浓度增加，△ψm下降、而线粒体膜蛋白表达显著升高。结论：ZGDHu-1能抑制HL-60细胞增殖和细胞活力，低浓度时诱导HL-60细胞向粒单系分化，高�AIM： To study the effect of N, N＇-di- （m-methylphenyi）-3,6-dimethyl- 1,4- dihydm- 1,2,4,5- tetrazine-1, 4-diearboamide （ ZGDHu-1 ） on proliferation inhibition, differentiation and apoptosis in HL-60 human leukemia cell line and explore its possible mechanism. METHODS ： Different eoneentration of ZGDHu- 1 and the different time of cultivate were used to treat HL-60 cell. The proliferation inhibition was analyzed by cell counting, alive cell count and MTF assay. Cell apoptosis was analyzed by cell morphology, DNA agamse gel eleetmphoresis, DNA content and Annexin-V/PI. The TdT-mediated dUTP nick end labeling （TUNEL） assay was used to quantitate the in situ cell apoptosis. The cell morphological analysis, expression of CD11b, CD13, CD14, CD64 and NBT reduction were performed to evaluate differentiation of HL-60 cells. The expressions of bcl-2, bax, Fas, P53 and mitochondrial membrane protein were analyzed by flow cytometry. While the mitochondrial transmembrance potential （△ψm） was labeled by dihydmrhodamin 123. RESULTS： ZGDHu-1 could inhibit HL-60 cell proliferation viability within a certain range of treating time and doses, with a 48 h IC50 of 180 ng·ml^-1 and 72 h of 180 ng· ml^-1 . A majority of HL-60 cells were arrested in G2/ M phase and a progressive decline in G0/1. The HL-60 cells apoposis was confirmed by type cell morphology, DNA fragment, sub-G1 phase, TUNEL and Annexin-V/ PI Labeling method with a time and dose related manner. The morphology of HL-60 cells cultured in the presence of 10- 100 ng·ml^-1 ZGDHu-1 for three days was more mature with higher positive rate of NBT and up-regulated expressions of CD11b, CD13, CD14 and CD64 than those of control. The expression of bax and Fas was increased, and bcl-2 was unchanged by the treatment of ZGDHu-1. ZGDHu-1 could increase the expression of mitochondrial membrane protein in a dose-dependent manner while the △ψm was reduced. CONCLUSION： ZGDHu- 1 can inhibit proliferation, induce differentiation and apoptosis of HL-60 cell

关 键 词：四嗪二甲酰胺 细胞株HL-60 细胞凋亡 细胞分化 

分 类 号：R965.2[医药卫生—药理学] R966[医药卫生—药学]