半导体量子点单克隆抗体荧光探针对牙乳头细胞内Smad4信号蛋白分子核移位过程的检测

The detection of the nucleation shifting process of smad4 signal protein in rat dental papillae cells with quantum dots-smad4 monoclonal antibody fluorescent probes

目的:制备半导体量子点-Smad4单克隆抗体荧光探针(Semiconductor quantumdots,QDS-Smad4),并用其对大鼠牙乳头细胞内Smad4蛋白分子在TGF-β1的刺激下发生的核移位过程进行检测。方法:①用化学偶连法制备水溶性的QDS-Smad4单抗荧光探针并纯化;②测定QDS-Smad4单抗荧光探针的吸收光谱、发射光谱并用激光共聚焦显微镜对QDS-Smad4单抗荧光探针的光学性质进行检测;③采用SP免疫组化法、QDS直接标记法、Smad4单抗直接标记法和QDS-Smad4单抗荧光探针直接免疫荧光成像法比较观察QDS-Smad4单抗荧光探针对大鼠牙乳头细胞内Smad4的特异性识别能力,并检测细胞内QDS-Smad4单抗荧光探针的光学性质;④在大鼠牙乳头细胞内加入TGF-β1,分别于加入前、加入后12h和24h,采用SP免疫组化法和QDS-Smad4单抗荧光探针直接免疫荧光成像法观察比较Smad4在细胞内发生核移位的动态变化。结果:半导体量子点与Smad4单抗通过共价结合形成稳定的QDS-Smad4单抗荧光探针,QDS-Smad4单抗荧光探针对大鼠牙乳头细胞内Smad4分子仍具有特异性的免疫识别能力,能成像显示Smad4所产生核移位的动态变化;QDS-Smad4单抗荧光探针仍具有QDS所具有的激发光谱宽,发射光谱窄,荧光度强,光化学稳定性好等光学特征。结论:半导体量子点和单抗共价结合形成分子探针后仍具有独特的光学性质和特异免疫识别能力,能长时间对细胞内蛋白质分子进行成像标记,这为半导体量子点用于可视化研究活细胞内蛋白质分子的运动和相互作用等过程提供了科学依据。

Objective：To prepare semiconductor quantum dots （QDs）-Smad4 monoclonal antibody Fluorescent probes ,and to detect nucleation shifting process of Smad4 signal protein in Rat dental papillae cells （RDPCs） stimulated by TGF-β 1 with the Fluorescent probes. Methods：（1）QDs were chemically modified with Smad4 proteins to prepare water soluble QDs-Smad4 monoclonal antibody Fluorescent probes which were purified after the preparation. （2）The properties of these probes were studied through absorption band analyses,fluorescence spectra,fluorescence spectrum analyses and confocal laser scanning fluorescence microscopy. （3）The location of Smad4 proteins in RDPCs was studied with SP anti-Smadd immunocytochemical method, QDs direct mark method, Smad4 monoclonal antibody direct mark method, and direct immunofluorescence imaging.And the properties of Quantum Dots-Smad4 monoclonal antibody Fluorescent probes in RDPCs were detected. （4）After RDPCs ware cultivated by TGF- β1 for 0 hours, 12 hours, and 14 hours,the dynamic nucleation shifting process of Smad4 signal protein in RDPCs was separately detected with SP anti-Smad4 immunocytochemical method and direct immunofluorescence imaging.Results：QDs and monoclonal antibody ware covalently bonded to form the fluorescent probes which could specifically and effectively recognize Smad4 proteins in RDPCs .It is to demonstrate the dynamic nucleation shifting process of Smad4 signal protein in RDPCs. These Fluorescent probes still had good properties,including broad excite spectra, narrow emission spectra, high fluorescence intensity, and photostability. Conclusion： QDs and monoclonal antibody could be covalently bonded to form the fluorescent probes with distinct optics character, special immunity recognition capability and the ability of imaging mark the protein in ceils for long time, which provides the scientific evidence that QDs can visually research the movement and interaction of protein in living cell.