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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 cobra venom (NNAV) on human erythroleukemia / adriamycin (K562 / ADM) cells and primary refractory acute myeloid leukemia cells. Methods: K562 / ADM cells and 10 cases of acute myeloid leukemia (AML) patients with primary primary refractory acute myeloid leukemia cells were studied. Different doses of NNAV were applied to the cells, using thiazolyl blue test The cell proliferation was measured by MTT assay. The expression of apoptosis-related protein Bcl2 was detected by immunocytochemistry. The apoptosis rate was detected by flow cytometry. The expression of Caspase-3 was detected by Western blotting. Flow cytometry was used to detect the effect of NNAV on cell cycle and to detect the expression of Cyclin-D in primary primary refractory acute myeloid leukemia cells. The above indexes were observed in Cyclin-D positive group and negative group Is there any difference between groups? Results: After different concentrations of NNAV treatment, the expression of Bcl-2 and Caspase-3 were up-regulated in both K562 / ADM cells and newly diagnosed acute myeloid leukemia cells. The cell proliferation was inhibited significantly and in a time-dependent manner Dose-dependent. Flow cytometry confirmed that NNAV at 0.8 mg / L and 1.0 mg / L both blocked the primary refractory cells in the G0 / G1 phase of the cell cycle while the cells in G2 / M phase decreased. In 10 cases of primary refractory acute myeloid leukemia cells, 7 cases showed positive expression of Cyclin-D, and 3 cases showed negative expression of Cyclin-D. However, there was no significant difference in the above indexes between Cyclin-D positive group and negative group. CONCLUSION: NNAV can significantly inhibit the proliferation of K562 / ADM cells and primary primary refractory AML cells in vitro. It can regulate the cell cycle arrest in G0 / G1 phase and decrease the cell content in G2 / M phase, suggesting that NNAV may interfere with Bcl- 2, Caspase-3 expression induced apoptosis, cell cycle progression may be cobra venom components of primary refractory AML cells to play a role in the inhibition of proliferation.