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目的探讨冬凌草甲素对白血病NB 4细胞的诱导凋亡作用及其机制。方法以不同质量浓度(8、16、24和32μm o l/L)的冬凌草甲素作用于体外培养的NB 4细胞。应用四氮唑蓝(M TT)比色法检测细胞生长抑制率,流式细胞术(FCM)检测细胞凋亡率;采用Hoechst 33258荧光染色法和琼脂糖凝胶电泳观察细胞凋亡;采用蛋白印迹(W estern b lot)和比色法测定细胞凋亡前后caspase-3表达水平及其活性的变化。结果冬凌草甲素(16μm o l/L以上浓度)对白血病NB 4细胞具有显著的增殖抑制及诱导凋亡作用,并且呈现出一定的时间-效应和剂量-效应关系。Hoechst 33258荧光染色观察到典型的核浓缩、核碎裂等细胞凋亡的形态学变化,琼脂糖凝胶电泳观察到细胞凋亡时的DNA“梯形”条带;W estern b lot检测结果表明32 kD a的caspase-3酶原被激活,出现20 kD a的亚单位活化片段,同时在细胞凋亡过程中,caspase-3活性显著增高。结论冬凌草甲素能够通过激活caspase-3的表达而诱导NB 4细胞发生凋亡,可为冬凌草甲素进一步应用于临床治疗急性白血病提供实验依据。
Objective To investigate the apoptosis-inducing effect of oridonin on leukemia NB 4 cells and its mechanism. Methods Oridonin at different concentrations (8, 16, 24, and 32 μmol/l) acted on NB 4 cells cultured in vitro. The inhibitory rate of cell growth was detected by MTT colorimetric assay, the apoptosis rate was detected by flow cytometry (FCM), and apoptosis was detected by Hoechst 33258 fluorescent staining and agarose gel electrophoresis. Western blotting (W estern b lot) and colorimetric assay were used to determine the changes of caspase-3 expression and its activity before and after apoptosis. RESULTS Oridonin (above 16 μmol/L) had significant inhibition of proliferation and induced apoptosis in leukemic NB 4 cells, and showed a certain time-effect and dose-response relationship. The morphological changes of cell apoptosis such as nuclear condensation and nuclear fragmentation were observed by Hoechst 33258 fluorescence staining. The “trapezoid” band of DNA was observed by agarose gel electrophoresis; the results of W estern b lot showed that 32 The kD a caspase-3 zymogen is activated and a 20 kD a subunit activation fragment appears, and the caspase-3 activity is significantly increased during apoptosis. Conclusion Oridonin can induce the apoptosis of NB 4 cells by activating the expression of caspase-3, which can provide experimental basis for the further application of oridonin in clinical treatment of acute leukemia.