摘要
采用基因质粒转染技术、荧光发射谱检测分析以及荧光共振能量转移(FRET)受体光漂白技术,首次在活细胞中实时检测中药蟾酥(Chan-Su,CS或bufonis venenum)诱导人肺腺癌(ASTC-a-1)细胞凋亡过程中caspase-3的活化特性.采用CCK-8(Cell Couneing Kit-8)技术检测发现,蟾酥对细胞的活性具有显著的抑制作用;蟾酥处理稳定表达FRET质粒SCAT3的人肺腺癌细胞后,在不同的时间检测活细胞中SCAT3的荧光光谱;利用共聚焦扫描荧光显微成像技术检测蟾酥处理后细胞的形态,从而进一步证实蟾酥诱导细胞凋亡.实验结果表明:a.蟾酥可以有效抑制人肺腺癌(ASTC-a-1)细胞的增殖活性并诱导细胞的死亡.蟾酥对细胞的抑制作用具有剂量依赖性;b.蟾酥处理细胞6h后能检测到明显的细胞凋亡小体,连续作用24h后细胞全部皱褶,并有部分细胞破碎;c.蟾酥作用细胞2h就能明显切割细胞内的SCAT3,细胞内SCAT3的切割程度随着蟾酥作用时间的延长而增加,24h内细胞内的SCAT3完全被切割.受体光漂白实验也证实了该结论,表明caspase-3参与调控了蟾酥诱导的细胞凋亡过程.
Chan-Su（CS, or bufonis venenum）, a traditional Chinese medicine, has many biological functions. It is mainly composed of bufotenidines, bufogenins, and etc. CS has been documented to possess functions against inflammation and cancer, and is widely employed as a therapeutic drug for many kinds of cancers in China. However, it is difficult to judge antitumor effect of agents derived from CS because of the following reasons： agents derived from CS are mixture, they are lack of observations of multiple centres, preparation process is lack of quality control standards and agents have many toxic or side effects at high dose. Apoptosis is a very important cellular event that plays a key role in pathogeny and therapy of many diseases. The mechanisms of the initiation and regulation of apoptosis are complex and diverse. Caspase family is closely connected with many apoptotic processes, while its member, caspase-3, being an important executive apoptotic molecule. To explore the inhibitory effect and mechanism of CS on human lung adenocarcinoma （ASTC-a-1）, gene plasmid transfection, fluorescence emission spectra and fluorescence resonance energy transfer （FRET） were used to study the caspase-3 activation during the CS-induced human lung adenocarcinoma （ASTC-a-1） cell apoptosis. CCK-8 was used to assay the inhibition of CS on the cells viability. The dynamical emission spectra of SCAT3 were performed inside living cells expressed stably with SCAT3 after CS treatment. The cell morphology was examined and photographed by phase microscope and confocal fluorescence scanning microscope. Experimental results showed that （1） CS inhibited dose-dependently the cells viability; （2） apoptotic body was observed in some cells 6 h after CS treatment, and most of cells were in shrinkage 24 h after CS treatment; （3） The SCAT3 inside living cells were cleaved by caspase-3 2 h after CS treatment, and most of the SCAT3 was cleaved 24 h after CS treatment, and the acceptor photobleaching experiments of SCAT3 also verified these results, implying that CS activated caspase-3 which induced cell apoptosis. Therefore, caspase-3 is involved in the CS-induced cell apoptosis.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2008年第1期85-90,共6页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(30670507,60378043,30470494)
广东省自然科学基金(015012)资助项目.~~
