论文部分内容阅读
目的:探讨冬凌草乙素(PON)对急性白血病NB4细胞的增殖抑制和诱导凋亡作用及其作用机制。方法:以不同浓度的PON(0～50μmol/L)作用于体外培养的NB4细胞0、24、48及72h,应用MTT法检测细胞生长抑制率,流式细胞术(FCM)检测细胞凋亡,Hoechst33258染色法检测细胞凋亡时的形态学变化。应用免疫印迹法检测Caspase-3及其裂解底物多聚(ADP-核糖)聚合酶PARP的表达水平,并对凋亡调节蛋白Bax、Bcl-2、Bak、Bad及Survivin的表达水平进行检测。结果:>20μmol/L的PON可显著抑制细胞的生长,并呈现出明显的量-效与时-效关系,FCM检测结果表明,>20μmol/L的PON作用48h后,凋亡细胞逐渐增多,在Hoechst染色后可见典型的细胞凋亡现象。蛋白质印迹法检测结果表明,药物作用48h后Caspase-3被活化出现17×103的亚单位,同时PARP被裂解出现89×103的亚单位片段。蛋白质印迹法检测结果显示,凋亡抑制蛋白Survivin的表达水平明显降低,促凋亡蛋白Bax的表达水平显著升高,而其他凋亡调节基因Bcl-2、Bak、Bad则无明显变化。结论:PON可以通过诱导白血病NB4细胞凋亡而发挥体外抗白血病作用,降低Survivin以及升高Bax的表达水平可能是PON诱导白血病细胞发生凋亡的重要作用机制之一。
Objective: To investigate the inhibitory effect of oridonin on proliferation and apoptosis of acute leukemia NB4 cells and its mechanism. METHODS: NB4 cells cultured in vitro were treated with different concentrations of PON (0-50 μmol / L) for 0, 24, 48 and 72 h. MTT assay was used to detect the cell growth inhibition rate. Flow cytometry (FCM) Hoechst33258 staining was used to detect morphological changes of apoptosis. Western blotting was used to detect the expression of PARP, an inhibitor of caspase-3 and its lytic substrate, and to detect the expressions of Bax, Bcl-2, Bak, Bad and Survivin. Results: PON at> 20μmol / L significantly inhibited cell growth and showed a dose-effect-time-effect relationship. FCM results showed that apoptotic cells increased after PON treatment for> 20μmol / L for 48h, Typical Hoechst staining shows signs of apoptosis. The results of Western blotting showed that Caspase-3 was activated to 17 × 103 subunits after 48 h treatment, and 89 × 103 subunit fragments were cleaved in PARP. The results of Western blotting showed that the expression of Survivin was significantly decreased and the expression of pro-apoptotic protein Bax was significantly increased, while the other apoptosis-regulated genes Bcl-2, Bak and Bad had no significant changes. CONCLUSION: PON can exert anti-leukemia effect in vitro by inducing apoptosis of leukemic NB4 cells. Decreasing the expression of Survivin and increasing Bax may be one of the important mechanisms of PON-induced apoptosis in leukemia cells.