摘要
目的研究华蟾素(cinobufagin)对人早幼粒白血病细胞株NB4的诱导凋亡作用,并探讨可能的分子机制。方法通过细胞计数观察华蟾素对细胞的生长抑制作用,荧光染色观察细胞凋亡的形态学改变,DNA凝胶电泳观察DNA的片段化改变,流式细胞术定量分析细胞凋亡。免疫荧光结合流式细胞术检测bcl-2表达、半定量RT-PCR检测Fas和FasL的表达。结果与对照组相比,1：100～1：25浓度的华蟾素能明显抑制NB4细胞生长。经华蟾素作用后,NB4细胞出现凋亡典型的形态学改变,核浓聚、核碎裂、凋亡小体形成；DNA电泳出现凋亡特有的“梯子”条带；流式细胞术也证实凋亡的存在。在经1：100、1：50、1：25浓度的华蟾素作用2d后,细胞凋亡率分别为12．49％、18．53％、37．76％。凋亡细胞的bd-2的表达下调,Fas表达上调,FasL表达无变化。结论华蟾素能抑制NB4生长并诱导凋亡,可能是通过下调bcl-2的表达,增加Fas的表达来实现。
Objective To investigate the proapoptotic effect of cinobufagin on NB4 cells and its mechanism. Methods The effect on growth inhibition of NB4 cells was evaluated by cell growth curve. The morphological assessment of apoptosis was performed by immunofluoresence staining. DNA fragment was visualized by agarose gel electrophoresis. The amount of apoptosis cells was measured by flow cytometry. The content of bcl-2 protein was analyzed by flow cytometry and the level of Fas and FasL mRNA were semi-quantified by RT-PCR. Rusults Comparing with the control group, the proliferation of NB4 cells was inhibited significantly by cinobufagin in the concentration of 1 ： 100- 1 ： 25. The morphological changes of typical apoptosis such as nuclear chromatin compacting, breaking and apoptotic bodies were observed. Agarose gel eletrophoresis of DNA revealed the apoptosis characteristic ladder pattern. Apoptosis was also confirmed by flow cytometry. The exposure of cinobufagin for 2 days in different dilution 1 ： 100 ,1 ： 50 and 1 ： 25,the apoptosis rate were 12.49% ,18. 53% and 37.76% .respectively. While cinobufagin induced apoptosis of NB4 cells, the expression of the bcl-2 protein increased and Fas mRNA decreased. FasL mRNA has no change. Conclusion Cinobufagin inhibits the proliferation and induces apoptosis of NB4 cells. Altering the expression of some genes involved in apoptosis, such as decreasing bob2 gene level and increasing Fas gene level, may be a mechanism of cinobufagin that induces apoptosis of NB4 cells.
出处
《肿瘤》
CAS
CSCD
北大核心
2005年第6期534-537,共4页
Tumor
基金
国家教育部骨干教师课题项目(编号:GGJS200013)
