【摘要】 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。更多还原

【Abstract】 Objective: To investigate the effects of naja naja atra venom(NNAV) on human erythroleukemia / adriamycin cells(K562/ADM cells) and primary culture cells of early onset refractory acute myelogenous leukemia.Methods: Ten cases of the cells mentionedwas studied.After having being treated with different concentrations of NNAV,cell proliferations were all oberved with the method of thiazolyl blue tetrazolium bromide(MTT).Expressions of Bcl-2,the apoptosis-related 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 caspase-3 expression.Furthermore,we detected cyclin D expressions of the primary culture cells of early onset refractory acute myelogenous leukemia alone,and above methods were used to find difference in the cyclin-D-positive expression group and the negative group.Results: After different concentrations of NNAV treated,both of the K562/ADM cells and primary culture cells of early onset refractory acute myelogenous leukemia resulted in a falled Bcl-2 and a raised caspase-3 expression,as well as cell proliferations obviously suppressed,and they were found to be time and dose-dependent.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 G0/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 results.However,it was of no significant difference in the cyclin-D-positive expression group and the negative group.Conclusion: This vitro study revealed that NNAV significantly inhibited cell proliferations of both K562/ADM cells and primary culture cells of early onset refractory acute myelogenous leukemia,and it also helped to regulate the cell cycle arrest in the G0/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 Bcl-2 and caspase-3 expression and induced them apoptosis.更多还原