在活细胞中应用FRET技术研究TGFβ/TβRI信号传导

转移生长因子β(TGFβ)信号传导通路参与调节细胞的增殖、分化、凋亡、细胞迁移等一系列细胞过程,与骨代谢疾病的发病机制密切相关.本研究根据荧光共振能量转移(FRET)技术原理,构建包含CFP-TβRI-YFP融合蛋白的TβRI生物传感器,转染293T细胞,观察转染效率.以TGFβ1为诱导剂,激活TGFβ/TβRI信号传导通路,在活细胞生理条件下,动态监测TβRI生物传感器的FRET效应.结果表明,成功构建了TβRI生物传感器,转染细胞效率达50%,在TGFβ1诱导刺激6min后,FRET效率增加并达到最大值,该过程经历9 min后,随时间的延长,FRET效率下降.研究结果表明:在活细胞生理条件下,TGFβ1/TβRI信号转导过程存在一定的时间特异性.

The Study of TGFβ/TβRI using FRET Technology in Living Cells

The interaction between transforming growth factor β (TGFβ) and TGFβ receptor I (TβRI) played a critical role of regulation of a variety of osteoporosis-related processes, such as cell proliferation, differentiation, apoptosis and migration. We constructed a TβRI biosensor in the form of a CFP-TβRI-YFP fusion protein. The TβRI biosensor construct was transfected into the 293T cells at about 50% of efficiency. Fluorescence resonance energy transfer (FRET）of TβRI biosensor were observed in live cells. Results showed that the FRET ratio of TβRI biosensor was maximized at 6 minutes when the ligand of TGFβ1 was added. The maximum level was kept about 9 minutes afterward,FRET ratios begin to decrease. Our study showed that the TGFβ 1/ TβRI signal transduction pathway might have a characteristic of time delay in living cells.