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目的观察四嗪二甲酰胺(ZGDHu-1)体外抑制白血病细胞系 SHI-1细胞增殖,诱导细胞分化和凋亡作用,并初步探讨其作用机制。方法将不同浓度 ZGDHu-1与 SHI-1细胞在体外共培养,用细胞计数、锥虫蓝染色、MTT 法、5′-溴-2′脱氧尿苷(Brdu)-ELISA 法观察 ZGDHu-1对 SHI-1细胞增殖的抑制作用;用细胞形态学、DNA 含量测定及细胞周期分析、膜联蛋白Ⅴ/碘化丙锭(Annexin Ⅴ/P1)双标记和 Hoechst 33258荧光染色等分析细胞凋亡。通过 NBT 试验、细胞表面抗原 CD11b、CD14、CD64检测和细胞形态学观察分化情况。用流式细胞术检测 ZGDHu-1诱导 SHI-1细胞分化和凋亡过程中 P38MAPK 和 STAT3磷酸化表达的变化。结果 ZGDHu-1能抑制 SHI-1细胞增殖和活力,呈现作用时间和剂量的量效关系,48和72h/C_(50)值分别为250ng/ml,85 ng/ml。ZGDHu-1作用于 SHI-1细胞后大部分细胞阻滞于 G_2/M 期,200ng/ml ZGDHu-1作用48h,SH-1细胞 G_2/M 期比例为(48.4±2.1)%;出现典型的细胞凋亡形态改变,DNA 片断化,检出亚 G_1峰,AnnexinⅤ/PI 和 Hoechst 33258荧光染色显示凋亡细胞的特征性改变。SHI-1细胞经2～50ng/ml ZGDHu-1作用3 d 后,细胞发生部分分化,NBT 阳性率增加,细胞表面 CD11b、CD14、CD64表达上调。ZGDHu-1作用 SHI-1细胞后磷酸化P38MAPK 表达增强,而磷酸化 STAT3表达降低。结论 ZGDHu-1能抑制 SHI-1细胞增殖和细胞活力.诱导细胞分化,促进细胞凋亡,其机制可能与 P38MAPK 的活化增强和 STAT3的活化抑制有关。
Objective To investigate the effect of ZGDHu-1 on proliferation, differentiation and apoptosis of leukemia cell line SHI-1 in vitro and its possible mechanism. Methods ZGDHu-1 cells were co-cultured with SHI-1 cells in different concentrations. The cell number, Trypan blue staining, MTT assay and Brdu-ELISA were used to observe the effect of ZGDHu-1 SHI-1 cells. The cell apoptosis was analyzed by cell morphology, DNA content determination and cell cycle analysis, annexin Ⅴ / PI double labeling and Hoechst 33258 fluorescence staining. NBT test, cell surface antigen CD11b, CD14, CD64 detection and cell morphology observed differentiation. Flow cytometry was used to detect the phosphorylation of P38MAPK and STAT3 during the differentiation and apoptosis of SHI-1 cells induced by ZGDHu-1. Results ZGDHu-1 could inhibit the proliferation and viability of SHI-1 cells. The dose-response relationship was observed between time and dose of ZGDHu-1. The 48 and 72h / C 50 values were 250ng / ml and 85ng / ml, respectively. ZGDHu-1 blocked most of the cells in G 2 / M phase after SHI-1 cells, while ZGDHu-1 cells treated with 200ng / ml ZGDHu-1 for 48h and G 2 / M phase in SH-1 cells 48.4 ± 2.1% Apoptotic morphological changes, DNA fragmentation, detected sub-G_1 peak, Annexin Ⅴ / PI and Hoechst 33258 fluorescence staining showed characteristic changes of apoptotic cells. After treated with 2 ~ 50ng / ml ZGDHu-1 for 3 days, SHI-1 cells partially differentiated and the positive rate of NBT increased. The expression of CD11b, CD14 and CD64 on the surface of SHI-1 cells was up-regulated. The effect of ZGDHu-1 on phosphorylated P38MAPK expression was enhanced in SHI-1 cells, while phosphorylated STAT3 expression was decreased. Conclusion ZGDHu-1 can inhibit SHI-1 cell proliferation and cell viability, induce cell differentiation and promote apoptosis, which may be related to the enhancement of P38MAPK activation and the inhibition of STAT3 activation.