四嗪二甲酰胺体外诱导NB4细胞增殖、凋亡与分化作用的研究  

Effects of N,N'-Di-(m-Methylphenyi)-3,6-Dimethyl-1,4-Dihydro-1,2,4,5-Tetrazine-1,4-Dicarboamide on Proliferation , Apoptosis and Differentiation of NB4 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] 吴建国[1] 刘建栋[1] 

机构地区：[1]浙江省人民医院中心实验室,杭州310014 [2]浙江工业大学药学院,杭州310014

出　　处：《中国实验血液学杂志》2006年第5期880-886,共7页Journal of Experimental Hematology

基　　金：浙江省科技厅资助项目;编号2003c33019

摘　　要：为了观察四嗪二甲酰胺(ZGDHu-1)对白血病细胞株NB4的增殖、分化和凋亡作用,并对其作用机制进行初步探讨。将不同浓度的ZGDHu-1与NB4细胞在体外培养,并以全反式维甲酸作阳性药物对照,观察ZGDHu-1对NB4细胞增殖抑制、凋亡和分化的作用。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。结果表明:ZGDHu-1对NB4细胞增殖和活力抑制呈时间和剂量的量效关系,48和72小时的IC50分别为450和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;细胞出现典型的形态改变,DNA片段化,亚G1峰检出并显著增加,Annexin-V/PI标记升高;Hoechst33258荧光染色后见凋亡细胞的特征性改变,这些均证实ZGDHu-1能诱导NB4细胞凋亡。NB4细胞经2-100ng/mlZGDHu-1作用3天后,细胞部分分化,NBT阳性率增加,细胞表面CD11b、CD13表达增高。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STAT3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,诱导细胞分化,促进细胞凋亡,其机制可能与bax表达上调、P38MAPK活化增强和端粒酶逆转录酶受抑有关。The purpose of this study was to explore the effect of N, N＇-di-（m-methylphenyi）-3,6-dimethyl-1,4-dihydro-1,2,4,5-tetrazine-1,4-dicarboamide （ZGDHu-1） on proliferation , differentiation and apoptosis in NB4 human leukemia cell line and its possible mechanism. Different concentrations of ZGDHu-1 and the different time of cultivation were used to treat NB4 cells. The proliferation inhibition of NB4 cells was analysed by cell counting, alive cell count , MTT assay. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin-V/PI and Hoechst33258 labeling method. The analysis of cell morphological change, expression of CD11 b, CD13 and NBT reduction were performed to evaluate the differentiation of NB4 cells. The expressions of bcl-2, bax and phosphorylated p38MAPK or STAT3 were detected by flow cytometry. While the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that ZGDHu-1 could inhibit NB4 cell proliferation viability within a certain range of treating time and does, IC50 values at 48 and 72 hours were 450 ng/ml and 200 ng/ml respectively. A majority of NB4 cells were arrested in G2/M phase and a progressive decline of cells was seen in G0/1. The NB4 cells apoptosis was confirmed by cell typical cell morphology, DNA fragments and sub-G1 phase peak as well as Hoechst33258 and Annexin-V/PI labeling method with a time- dose-related manner. The morphology of NB4 cells cultured in the presence of 2 - 100 ng/ml ZGDHu-1 for three days was more mature with higher NBT positivity and expressions of CDllb and CD13 than those in control. The expression of phosphor-p38MAPK and bax was increased while phosphor-STAT3 and bcl-2 were unchanged by the treatment of ZGDHu-1. ZGDHu-1 could decrease the expression of hTERT-mRNA in a dose-dependent manner, It is concluded that ZGDHu-1 can inhibit proliferation, induce differentiation and apoptosis of NB4 cells. The mechanism may be associated with up-regulation o

关 键 词：四嗪二甲酰胺 NB4细胞株 有丝分裂原激活蛋白激酶 BAX/BCL-2 端粒酶逆转录酶 白血病 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学] R733.7[医药卫生—基础医学]