单PC12细胞神经活性分子释放的原位电化学/拉曼光谱研究

The study of neurotransmitter exocytosis and the correlation between different neuroactive molecules at the single living cell level is crucial to the understanding of the laws governing the operation of the whole nervous system. There exist several basic scientific questions in the electrochemical study of single living PC12 cells, including the release behaviors of acetylcholine and their correlation with those of catecholamine and other messengers, the in situ real time probing of minute electrochemically active or non-active species released in accompany with the quantal release of catecholamine, etc. Aiming at solving these issues, we proposed two strategies to allow simultaneous real time multiplex analysis of the release behaviors of a variety of neural active signaling molecules of PC12 cells and their correlations. One is to design ultramicro- and nano- composite electrodes for direct electrochemical detection on single cell. The other is to combine Raman spectroscopy with single cell electrochemical detection system by constructing highly SERS-active ultramicroelectrode. We will also further construct multi-cell models based on patterned substrate to probe the signals reflecting the interactions between adjacent cells. We expect to provide important insights into the related diseases.

单活细胞水平上的神经递质的胞吐释放以及神经活性物质间的相关性研究对了解整个神经系统运行规律有着至关重要的作用。针对单活PC12细胞胞吐作用的电化学研究中存在的基本科学问题，包括乙酰胆碱的释放规律及其与主要神经递质儿茶酚胺释放的关联性、儿茶酚胺胞吐释放过程中存在的少量其它电化学活性/非电化学活性物质的实时共检测等问题，本项目提出通过发展基于超微及纳米复合电极的电化学检测技术和发展电化学与拉曼光谱联用技术应用于PC12细胞的多种神经活性信号分子包括儿茶酚胺、乙酰胆碱、一氧化氮、过氧化氢等释放的实时原位共检测，全面考察PC12细胞多种神经活性物种的释放规律及其相关性；并进一步通过模板法建立多级结构细胞模型，考察细胞间信号传导对胞吐作用的影响。项目研究结果可望为相关疾病病理研究提供理论和实验依据。

神经递质的囊泡释放和传输是维持神经系统功能及其与许多其它组织相互作用的中心机制。因此对于囊泡释放的实时监测具有重要的意义并引起研究者的高度兴趣。本项目以PC12细胞儿茶酚胺胞吐释放为模型体系，主要在致密核心大囊泡胞吐作用机理、液/液界面电极及其在单细胞体系的应用、拉曼光谱的引入及联用系统建设等多方面开展了一系列开拓性的研究工作。目前主要在PC12细胞胞吐作用机理（多巴胺和去甲肾上腺素共释放）方面取得了重要进展，在单细胞水平给出了胞吐作用的“部分释放”机理的直接证据，并提出了“部分释放”机理的理论模型，相关工作发表于JECS；在有关方法学和检测系统两方面工作在取得一定进展的同时也遇到一些瓶颈问题，仍有待进一步的深入探索。