中华眼镜蛇毒对K562/ADM和难治性急性髓性白血病细胞作用研究

Effect of naja naja actra venom on K562/ADM and refractory acute myeloid leukemia cells

目的:探讨中华眼镜蛇毒(NNAV)对人红白血病/阿霉素细胞(K562/ADM细胞)及原代难治性急性髓性白血病细胞的作用。方法:以K562/ADM细胞及10例急性髓性白血病(AML)患者的原代初发难治性急性髓性白血病细胞为研究对象进行实验,应用不同剂量的NNAV作用于细胞后,采用噻唑蓝实验法(MTT)测定细胞增殖情况,免疫细胞化学法检测凋亡相关蛋白Bcl2表达,流式细胞仪测定凋亡率,蛋白质印迹法检测Caspase-3表达。流式细胞仪检测NNAV对细胞周期的影响,检测原代初发难治性急性髓性白血病细胞细胞周期蛋白D(Cyclin-D)的表达情况;观察以上指标在Cyclin-D阳性表达组和阴性表达组有无差异。结果:经过不同浓度的NNAV处理后,无论是K562/ADM细胞还是初发难治性急性髓性白血病细胞,Bcl-2表达下调,Caspase-3表达上调;细胞增殖抑制明显,并具有时间依赖性及剂量依赖性。流式细胞仪检测证实0.8 mg/L和1.0 mg/L的NNAV均能够使原代初发难治性细胞阻滞于细胞周期的G0/G1期,而处于G2/M期的细胞含量减少。10例原发难治性急性髓性白血病细胞中Cyclin-D表达阳性7例,Cyclin-D表达阴性3例,但以上指标在Cyclin-D阳性表达组和阴性表达组无显著差异。结论:NNAV在体外可明显抑制K562/ADM细胞和原代初发难治性AML细胞增殖;调节细胞周期停滞于G0/G1期,减少G2/M期的细胞含量,提示NNAV有可能通过干预Bcl-2、Caspase-3表达而诱导细胞凋亡,细胞周期进程可能是中华眼镜蛇毒组分对原发难治性AML细胞的发挥增殖抑制作用的机制。

Objective： To investigate the effects of naja naja atra venom （NNAV） on human erythroleukemla adriamycin cells （ K562/ADM ceils） and primary culture cells of early onset refractory acute myelogenous leuke mia. Methods： Ten cases of the ceils mentionedwas studied. After having being treated with different concentra tions of NNAV, cell proliferations were all oberved with the method of thiazolyl blue tetrazolium bromide （MTT） . Expressions of Bcl2, the apoptosisrelated proteins was studied by the immunocytochemistry assay. Besides, flow cytometry was used to measure their apoptosis rate and effects of NNAV on cell cycle, and Western blot was for caspase3 expression. Furthermore, we detected cyclin D expressions of the primary culture cells of early onset re fractory acute myelogenous leukemia alone, and above methods were used to find difference in the cyclinDpositive expression group and the negative group. Results： After different concentrations of NNAV treated, both of theK562/ADM cells and primary culture cells of early onset refractory acute myelogenous leukemia resulted in a falled Bcl2 and a raised caspase3 expression, as well as cell proliferations obviously suppressed, and they were found to be time and dosedependent. NNAV with concentration of 0. 8 mg/L and 1.0 mg/L could made cell cycle of the primary culture cells of early onset refractory acute myelogenous leukemia arrest in the GO/G1 phase, while the cell contents decreased at the G2 / M phase. For the primary culture cells of early onset refractory acute myelogenous leukemia alone, we observed that 7 of 10 were with CyclinD positive expression, the other 3 were with negative re suits. However, it was of no significant difference in the cyclinDpositive expression group and the negative group. Conclusion： This vitro study revealed that NNAV significantly inhibited cell proliferations of both K562/ADM ceils and primary culture cells of early onset refractory acute myelogenous leukemia, and it also helped to regulate the cell cycle arrest in the GO/G1 phase, reduced the cell content in the G2 / M phase. Our experiment suggested that NNAV possibly inhibited the cell proliferations of primary culture cells of early onset refractory acute myelogenous leukemia through the intervention of Bcl2 and caspase3 expression and induced them apoptosis.