Chen Qin-Fen, Chen Zi, Li Pei, Fan Xue-Liang, Zhang Shao-Feng, Yuan Yan, Ding Tian-Ling, Xie Yi
Departmemt of Hematology, Huashan Hospital, Fudan University, Shanghai 200040, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2008 Feb;16(1):74-7.
The aim of this study was to investigate the efficacy of diacetyl hexamethylene diamine (CAHB) for patients with high risk myelodysplastic syndrome (MDS), and to explore the effect of CAHB on HL-60 cells in vitro and its possible mechanism. 8 patients with high risk MDS were treated with CAHB by continuous intravenous infusion for 10 days, and repeated once after an interval of 28 days. The count of the granulo- and mono-blasts in bone marrow (BM) aspirate was measured before and after treatment. HL-60 cells were treated with different concentrations of CAHB for 72 hours in vitro. The inhibitory effect of CAHB on proliferation of HL-60 cells in vitro was measured by MTT assay. Differentiation of HL-60 cells was detected by the changes of CD11b and CD14 expression on cell surface. Apoptosis of HL-60 cells was detected by double staining of Annexin V and PI. The cell cycle distribution change of HL-60 cells was analyzed by flow-cytometry. The results indicated that the granulo- and mono-blasts in BM decreased in all the 8 patients after CAHB treatment. The main side effect of CAHB on hematological system was thrombocytopenia. After being treated with 1, 2, 3, 4 mmol/L CAHB for 72 hours in vitro, the result of MTT assay showed the inhibitory effect of CAHB on the proliferation of HL-60 cells in dose-dependent manner. After being treated manner 1, 2, 3, 4 mmol/L CAHB for 72 hours, the CD11b positive HL-60 cells were 22.39+/-3.97%, 33.12+/-4.46%, 49.25+/-5.27%, 78.05+/-5.66%, respectively, which were significantly different from the control group (CD11b positive HL-60 cells was 5.89+/-2.94%) (p<0.01). The CD14 expression was negative in all the 5 groups. These results suggested that CAHB could induce HL-60 cells to differentiate into mature granulocytes, and the effect of CAHB appeared in dose-dependent manner. After being treated for 72 hours by 1, 2, 3, 4 mmol/L CAHB, the apoptotic cells (Annexin V(+)/PI(-) cells) increased mildly, which suggested that CAHB only weakly induces HL-60 cells to apoptosis at the concentration of 1 to 4 mmol/L. Along with the concentration increase of CAHB, the ratio of cells in G(0)/G(1) phase increased, and ratio of cells in S phase and G(2)/M phase decreased correspondingly, it indicated that CAHB could arrest HL-60 cells in G(0)/G(1) phase in a dose-dependent manner. It is concluded that induction of cell differentiation may be the primary effect of CAHB on MDS. Cell cycle arrest may be essential to the effect of CAHB as well. Side effect of CAHB on platelet count may correlated with its inhibitory effect on hematopoiesis.
本研究旨在探讨二乙酰己二胺（CAHB）对高危骨髓增生异常综合征（MDS）患者的疗效，并在体外研究CAHB对HL-60细胞的作用及其可能机制。8例高危MDS患者接受CAHB持续静脉输注治疗10天，间隔28天后重复治疗一次。治疗前后检测骨髓穿刺液中粒系和单核系原始细胞计数。体外将HL-60细胞用不同浓度的CAHB处理72小时。采用MTT法检测CAHB对体外HL-60细胞增殖的抑制作用。通过细胞表面CD11b和CD14表达的变化检测HL-60细胞的分化。采用Annexin V和PI双染法检测HL-60细胞的凋亡。通过流式细胞术分析HL-60细胞的细胞周期分布变化。结果显示，8例患者经CAHB治疗后骨髓中粒系和单核系原始细胞均减少。CAHB对血液系统的主要副作用是血小板减少。体外经1、2、3、4 mmol/L CAHB处理72小时后，MTT检测结果显示CAHB对HL-60细胞增殖的抑制作用呈剂量依赖性。经1、2、3、4 mmol/L CAHB处理72小时后，CD11b阳性的HL-60细胞分别为22.39±3.97%、33.12±4.46%、49.25±5.27%、78.05±5.66%，与对照组（CD11b阳性的HL-60细胞为5.89±2.94%）相比差异有统计学意义（p<0.01）。5组中CD14表达均为阴性。这些结果提示CAHB可诱导HL-60细胞分化为成熟粒细胞，且作用呈剂量依赖性。经1、2、3、4 mmol/L CAHB处理72小时后，凋亡细胞（Annexin V(+)/PI(-)细胞）轻度增加，提示CAHB在1至4 mmol/L浓度下仅微弱诱导HL-60细胞凋亡。随着CAHB浓度增加，G(0)/G(1)期细胞比例增加，S期和G(2)/M期细胞比例相应降低，表明CAHB可呈剂量依赖性地将HL-60细胞阻滞于G(0)/G(1)期。结论是诱导细胞分化可能是CAHB对MDS的主要作用。细胞周期阻滞可能也是CAHB发挥作用的关键。CAHB对血小板计数的副作用可能与其对造血的抑制作用有关。
