利用FRET技术实时检测Bid蛋白在TNF-α诱导PC12细胞凋亡过程中的作用

Real-time detection of the role of Bid protein in TNF-α-induced apoptosis of PC12 cells by usingFRET

】目的在活细胞中实时检测TNF-α诱导PCI2细胞凋亡过程中Bid蛋白的作用并探讨PCI2细胞凋亡的信号通路。方法利用基于荧光共振能量转移（nuoreseence resonance energyt ransfer，FRET）原理设计的荧光探针pFRET-Bid（YFP-Bid-CFP）转染PC12细胞后，在激光共聚焦显微镜下实时检测Bid蛋白的切割情况，并采用专用软件进行数据分析。结果TNF-α诱导细胞凋亡的过程中，在短时间内（3h）就凋亡的细胞中观测不到bid的切割，而在8h后凋亡的细胞中却观测到了bid的切割。结论通过分析实验结果推测不同时间凋亡的细胞经过的信号通路是不同的，一个是不通过线粒体的较快途径，一个是需要通过线粒体的较慢途径。

Objective To real-time detect the role of Bid protein in TNF-ct induced-apoptosis in PC12 cells and to explore the signaling pathways of apoptosis in living cells. Methods A pFRET-Bid probe, designed based on the principle on fluorescence resonance energy transfer （ FRET ）, was used to transfect PC12 cells, and the cutting of Bid protein was real-time detected by a laser scanning confocal microscope. The data wet~ analyzed using a special software. Results Bid cutting was observed in the apoptotic cells 8h after induction with TNF-ct but not with in a short period of time （ 3 h ）. Conelusions By analyzing the experimental results＇, we presume apoptotic cells at different times have different signaling pathways, one is the faster non-mitochondrial pathway, and the other is the slower mitochondrial pathway.