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32μmol/L)的冬凌草甲素作用于体外培养的NB4细胞.应用四氮唑蓝(MTT)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(Western blot)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(16μmol/L以上浓度)对白血病NB4细胞具有显著的增殖抑制及诱导凋亡作用, 并且呈现出-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;Western blot检测结果表明32kDa的caspase-3酶原被激活,出现20kDa的亚单位活化片段,同时在细胞凋亡过程中,casp-ase-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NB4细胞发生凋亡,可为冬凌草甲素进-步应用于临床治疗急性白血病提供实验依据.

Objective To investigate the mechanisms of oridonin inducing apoptosis on acute leukeamia NB4 cells and its mechanism.Metho-ds NB4 cells in culture mediumin vitro were given with different concentrations(8,16,24,and 32/1mol/L)of oridonin.The inhi-bitory rate of the cells was measured by MTT assay,cell apoptotic rate was detected by flow cytometry(FCM),morphology of apoptosis was observed by Hoechst 33258 fluorescence staining,DNA fragmentation was assayed by agarose gel electrophoresis,caspase-3 expression was detected by Western blotting,and caspase-3 activity was assayed with colorimetric assay kit before and after apoptosis occurred. Results Oridonin(over 16 μmol/L)could inhibit the growth of NB4 cells and cause apoptosis significantly,the suppression was both in a time-and dose-dependent manner.Marked changes of apoptosis includifig condensation of ehromatin and nuclear fragmentation were observed very clearly by Hoechst 33258 fluorescence staining and a characteristic"ladder"of DNA fragments was elicited by agarose gel electrophoresis;Western blot analysis revealed that caspase-3 was activated by the lOSS of caspase-3 proenzyme(32 kDa)and the appea-rance of its 20 kDa subunit,and that along with the apoptotic process caspase-3 activity was increased concurrently.Conclusion Oridonin can induce apoptosis in NB4 cells via activation of caspase-3.These results wilI provide laboratory evidence for the clinical treatment of acute leukemia with oridonin.

国家自然科学基金资助项目(30300312)