太白银莲花皂苷-A诱导人胶质瘤U87细胞凋亡及其机制研究

目的探讨太白银莲花皂苷A诱导胶质瘤U87细胞株细胞凋亡作用及机制。方法以四甲基偶氮唑蓝(MTT)比色法检测太白银莲花皂苷A对U87细胞和VEC304细胞存活率的影响;Hoechst 33342核染色后荧光显微镜下观察细胞核形态学变化;透射电子显微镜观察太白银莲花皂苷A处理后U87细胞超微结构;流式细胞仪AnnexinV/IP双染检测太白银莲花皂苷A处理后U87细胞凋亡比率;免疫印迹法(Western blot)检测致凋亡分子Fas、Fas-L、Pro-Caspase8、Pro-Caspase3、Bcl-2、Bax等表达情况。结果极低浓度(<5 mg/L)下太白银莲花皂苷A处理后U87细胞生存率明显下降,同比正常EVC304细胞生存率影响不显著;Hoechst 33342核染色及透射电镜下均可见典型细胞凋亡形态学变化;流式细胞仪分析显示低浓度太白银莲花皂苷A(1.75 mg/L IC50)处理后U87细胞系早期凋亡细胞比率随时间递增,呈明显时间依赖性;免疫印迹实验发现太白银莲花皂苷A处理U87细胞6,12,24 h后细胞Fas及Fas-L表达递增,Pro-Caspase8、Pro-Caspase3递减,活化Caspase-3片段12 h达到峰值,Bcl-2表达差别不显著,Bax表达未测出。结论太白银莲花皂苷A能够诱导U87细胞凋亡,并且其机制与Fas介导的表面受体凋亡通路有关。

A novel triterpenoid taipaienoside A induces human glioma U87ceils apoptosis and the mechanism research

Objective It is to investigate the potential apoptosis-inducing effect and the mechanisms of a novel triterpenoid-taipaienoside A on human glioma U87 cell lines.Methods The viability of U87 and VEC304 cells treated by taipaienoside A was detected by methyl thiazolyl tetrazolium(MTT)chromatometry;Cytomorphology changes was observed on fluorescence microscope after staining cell chromosome by Hoechst33342.Transmission electron microscope(TEM) was also applied to reveal ultrastructure of U87 cells treated by taipaienoside A.The proportions of apoptotic cells were evaluated by flow cytometric Annexin V/IP staining analysis.To probe the apoptotic mechanisms induced by taipaienoside A,expressions of Fas,Fas-L,Pro-caspase8,Pro-caspase3,Bcl-2,and Bax in U87 cells were examined via Western Blot analysis.Results The viability of U87 cells declined significantly after treated with taipaienoside A at extremely low concentrations(<5 mg/L),and normal cells VEC304 were not influenced significantly in compare;Typical apoptosis morphology changes were observed both in Hoechst33342 staining assay and TEM;The proportions of apoptosis in U87 cells at early stage increased time-dependently after treated with taipaienoside A(1.75 mg/L) for indicate time in Annexin V/PI staining assay;Fas and Fas-L expression was up-regulated in U87 cells after treated with taipaienoside A for 6,12 or 24h,accompanied with Pro-caspase8 and Pro-caspase3 decline,cleaved caspase3 peaking at 12h,Bcl-2 expressing no significant difference,Bax undetectable in western blot assays.Conclusion Taipaienoside A possesses potent ability of inducing U87 cells apoptosis,and the mechanisms was at least involved by Fas/FasL-associated cascade-dependent apoptotic mechanisms.