四嗪二甲酰胺体外诱导NB4细胞增殖、凋亡及分化作用探究.docVIP

四嗪二甲酰胺体外诱导NB4细胞增殖、凋亡及分化作用探究 　 作者：周永列，吕亚萍，胡惟孝，邱莲女，王文松，吴建国， 刘建栋 【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200 ng/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2-100 ng/ml ZGDHu-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。 【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 　　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 　　　AbstractThe 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 CD11b, 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 ZGD