蟾酥灵诱导细胞内caspase-3活化特性荧光分析

Analyzing caspase-3 activation induced by bufalin using fluorescence techniques in single living cell

采用多光子激发荧光光谱和寿命成像显微技术,在单个活细胞内实时检测蟾酥灵(bufalin,Bu)诱导人类肺腺癌(ASTC-a-1)细胞死亡过程中caspase-3的活化特性.利用CCK-8(Cell Counting Kit-8)检测蟾酥灵对人类肺腺癌细胞活性的抑制效应.蟾酥灵处理稳定表达FRET质粒SCAT3的人类肺腺癌后,在不同时间点检测单个活细胞中SCAT3的多光子激发荧光光谱及其供体ECFP的荧光寿命,从而检测bufa-lin诱导细胞凋亡过程中caspase-3活化特性.实验结果表明:蟾酥灵以浓度依赖性的方式显著地抑制人类肺腺癌细胞的生长;蟾酥灵处理细胞24h后,细胞内SCAT3未被切割,而蟾酥灵作用细胞48h后SCAT3被切割.说明caspase-3参与调控蟾酥灵诱导人类肺腺癌细胞凋亡的过程.

Caspase-3 activation was analyzed inside single living cells during bufalin-induced human lung adenocarcinoma （ ASTC-a-1 ） cell apoptosis by using multi-photon excitation spectra and fluorescence lifetime imaging microscopy （FLIM）. CCK-8 was used to assay the inhibition of bufalin on the ceils＇ viability. The dynamical emission spectra of SCAT3 which is a fluorescence resonance energy transfer （FRET） plasmid, and fluorescence lifetime of ECFP were performed inside single living cells, stably expressing SCAT3 after bufalin treatment. Compared with controls, SCAT3 did not change after 24 h, but was cleaved after 48 h, which were verified by the changes of fluorescence spectra of SCAT3 and fluorescence lifetime of ECFP. Our data shows that the cells＇ growth is significantly inhibited by bufalin in a dose-dependent manner; caspase-3 is involved in the bufalin-induced cell death.