【作者】 周永列； 吕亚萍； 胡惟孝； 邱莲女； 王文松； 吴建国； 刘建栋；

【Author】 Zhou Yong-Lie, LV Ya-Ping, Hu wei-xiao, QIU Lian-Nu , WANG Wen-Song, Wujian-guo ,Uu Jian-dong Central Laboratory,Zhejiang Provincial People’s Hospital,Hangzhou 310014 China , Colleage of Pharmaceutical Sciences,Zhejiang University of Technology Hangzhou 310014,China

【机构】 浙江省人民医院中心实验室； 浙江工业大学药学院；

【摘要】 目的:观察四嗪二甲酰胺(ZGDHu-1)体外抑制白血病细胞株NB4增殖,诱导细胞分化争凋亡作用,并对其作用机制进行初步探讨．方法:将不同浓度的ZGDHu-1与NB4细胞在体外培养,并以全反式雏甲酸作阳性药物对照,用细胞计数、台盼蓝染色、MTT比色法观察ZGDHu-1 对NB4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Hoechst33258荧光染色等分析细胞凋亡．通过NBT试验、细胞表面抗原 CD11b、CD13和细胞形态学检测ZGDHu-1对NB4细胞的分化作用．用流式细胞术检测ZGDHu-1诱导NB4细胞分化和调亡过程中bcl-2和bax的表达变化,以及P38MAPK和Stat3磷酸化水平, 同时利用实时荧光PCR定量检测端粒酶hTERT-mRNA表达的变化．结果:ZGDHu-1呈现作用时间和剂量的量效性抑制NB4细胞增殖和活力,48h、72h的IC50分别为450、200ng/ml．NB4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,亚G1峰检出并显著增加,Annexin-V/P工标记升高,Hoechst33258荧光染色后的凋亡细胞的特征性改变等均证实ZGDHu-1能诱导NB4细胞凋亡．NB4细胞经2-100ng/ml 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表达增加．ZGDHu-1作用NB4 细胞后,bcl-2表达无变化,但bax表达显著增加,bax/bcl-2的比值升高,磷酸化P38MAPK表达增强,而磷酸化Star3表达无变化,端粒酶hTERT-mRNA的表达随ZGDHu-1作用的浓度增加而显著下调．结论:ZGDHu-1能抑制NB4细胞增殖和细胞活力,诱导细胞分化,促进细胞凋亡,其机制可能bax表达上调,P38MAPK的活化增强和抑制端粒酶逆转录酶有关。更多还原

【Abstract】 Objective To study the effect of N,N’ -di-(m-methylphenyi)-3,6-dimethyl-1,4- dmydro-1,2,4,5-tetrazine-1,4-dicarboamide(ZGDHu-1) on proliferation inhibition ,differentiation and apoptosis in NB4 human leukemia cell line and explore its possible mechanism. Method Different concentration of ZGDHu-1 and the different time of cultivate were used to treat NB4 cell. The proliferation inhibition was analysed by cell counting, alive cell count , MTT assay. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin-V/PI and Hoechst33258 labeling method .The cell morphological analysis,expression of CD11b,CD13 and NBT reduction were performed to evaluated differentiation of NB4 cells. The expressions of bcl-2,bax and phosphorylated p38MAPK or STAT3 were analysed by flow cytometry. While the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. Results ZGDHu-1 can inhibit NB4 cell proliferation viability within a certain range of treating time and does,with a 48 h IC50 of 450ng/mland 72h of 200ng/mlo a majority of NB4 cells were arrested in G2/M phase and a progressive decline in G0/1 . The NB4 cells apoposis was comfirmed by type cell morphology and DNA fragment and sub-G1 phase and Hoechst33258 and Annexin-V/PI Labeling method with a time and does related manner. The morphology of NB4 cells cultured in the presence of 2- 100ng/ml ZGDHu-1 for three days was more mature with higher NBT positivity and up-regulated expressions of CD11b,CD13 than that of control. The expression of phosphor-p38MAPK and bax was increased and phosphor-STAT3 and bcl-2 was unchanged by the treatment of ZGDHu-1. ZGDHu-1 could decrease the expression of hTERT-mRNA in a dose-dependent manner. Conclusion ZGDHu-1 can inhibit proliferation, induced differentiation and apoptosis of NB4 cells. The mechanism may associate with its up-regulation of phosphor-p38MAPK,bax and down-regulation hTERT-mRNA.更多还原