利用荧光共振能量转移技术在活细胞内研究顺铂诱导的凋亡信号通路

Using FRET Technique to Investigate The Apoptotic Mechanism Induced by Cisplatin in Living Cells

为在活细胞内探讨顺铂诱导的凋亡通路.实验样品经顺铂处理后,应用基于荧光共振能量转移(FRET)原理设计的荧光探针pFRET-Bid和pSCAT-3来检测Bid切割和Capase-3活化的动态变化,同时,利用荧光成像在亚细胞水平对Bid转位线粒体的动力学特征进行了实时分析.结果表明:在顺铂诱导的细胞凋亡过程中,Bid切割发生在药物刺激后4～5h,历时(120±20)min.Bid切割活化后即从胞浆内转位到线粒体,历时(90±15)min.在凋亡后期,可以明显检测到Caspase-3的激活.研究表明,应用FRET及荧光成像技术,可以在活细胞内实时、直观、可视地研究顺铂诱导的细胞凋亡过程,从而客观地反映了Bid、Caspase-3等蛋白质分子在该凋亡信号通路中的动态行为及时空传递特性.

Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cell. However, the signal transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully understood. Using fluorescence resonance energy transfer （FRET） technique, the molecular mechanism of cisplatin-induced apoptosis in living human lung adenocarcinoma cells （ASTC-a-1） were investigated. After cisplatin treatment, the recombinant pFRET-Bid and pSCAT-3 probes were used to determine the kinetics of Bid cleavage and Caspase-3 activation, respectively. The fluorescence probes Bid-CFP and DsRed-Mit were also used to detect the spatial and temporal changes of Bid in real-time in sub-cell level. The results showed that a cleavage of the Bid-FRET probe occurring at about 4-5 h after treated with 20 μmol/L cisplatin. Cleavage of the Bid-FRET probe coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h. At the anaphase of cell apoptosis, Caspase-3 was activated obviously as detected by FRET and Western blotting techniques. Using real-time single-cell analysis, it was observed the kinetics of Bid and Caspase-3 activation for the first time in living cells during cisplatin-induced apoptosis.