消癌平诱导人肺腺癌(ASTC-a-1)细胞内caspase-3活化的荧光光谱分析  

Fluorescence Emission Analysis of Caspase-3 Activation Induced by Xiao-Ai-Ping(XAP) inside Living Human Lung Adenocarcinoma Cells

作　　者：陈同生[1] 王小平[2] 王治平[3] 王龙祥[1] 王会营[1] 邢达[1] 

机构地区：[1]华南师范大学激光生命科学研究所 暨激光生命科学教育部重点实验室,广东广州510631 [2]暨南大学附属第一医院麻醉科,广东广州510632 [3]广州中医药大学科技产业园有限公司,广东广州510445

出　　处：《光谱学与光谱分析》2008年第6期1327-1331,共5页Spectroscopy and Spectral Analysis

基　　金：国家自然科学基金项目(30670507;60378043;30470494);中国博士后科学基金项目(2005037169)资助

摘　　要：采用CCK-8技术检测发现传统中药消癌平(XAP)对人肺腺癌(ASTC-a-1)细胞的增殖活性具有显著的抑制作用。利用共聚焦扫描荧光显微成像、荧光共振能量转移(FRET)及其受体光漂白技术证实了基于FRET技术构建的SCAT3质粒在ASTC-a-1细胞中的稳定表达。将消癌平加入细胞培养液中培育细胞,并在不同的时间检测活细胞内SCAT3的荧光发射光谱,从而监测细胞中caspase-3的活化状态。实验结果表明:(1)消癌平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,消癌平对细胞的抑制作用具有剂量依赖性。(2)消癌平处理细胞72 h后,细胞内大量的SCAT3被切割,表明细胞内caspase-3的活化水平明显升高。(3)将含消癌平的细胞培养液与细胞共同培养24 h,然后采用没有消癌平的细胞培养液培养细胞48和72 h后,细胞内SCAT3的光谱没有明显改变,表明消癌平作用细胞24 h内没有显著激活细胞内的caspase-3。The CCK-8 was used to measure the inhibition effect of Xiao-Ai-Ping （XAP）, a traditional medicine, on the human lung adenocarcinoma （ASTC-a-1） cells viability. The ASTC-a-1 cells expressing stably with SCAT3, a fluorescence resonance energy transfer （FRET） plasmid based on the green fluorescent protein mutants （GFPs）, was verified using confocal fluorescence scanning microscopy imaging, fluorescence emission spectra and FRET acceptor photobleaching techniques. The caspase-3 activation can be monitored by the fluorescence emission spectra of SCAT3 inside living cells. The cells expressing stably with SCAT3 were cultured with XAP for 96 hours, and the fluorescence emission spectra of the SCAT3 inside living cells were measured at the time of 0, 24, 72, and 96 hours, respectively. Experimental results showed that： （1）XAP inhibited obviously the proliferation of ASTC-a-1 cells and induced the cell death. The inhibition of XAP on the cells was dose-dependent; （2）the SCAT3 inside living cells was cleaved completely 72 hours after the XAP treatment, implying that a great deal of pro-caspase-3 was activated by XAP; （3）24 hours after XAP treatment, the emission spectra of SCAT3 inside living cells cultured in DMEM without XAP for 48 arid 72 hours did not change greatly, implying that XAP did not activate obviously caspase-3 within 24 hours.

关 键 词：消癌平 荧光光谱 caspae-3 荧光共振能量转移(FRET) 激光共聚焦荧光显微镜 

分 类 号：Q631[生物学—生物物理学]