培养大鼠皮层神经元NMDA受体蛋白单分子原子力显微镜定位研究

为在纳米尺度对 NMDA受体蛋白分子进行神经细胞膜表面原位定位和探讨原子力显微镜在生物单分子操纵和调控中的应用 ,本研究应用原子力显微镜分别对分布在云母表面的膜 NMDA受体蛋白分子标记物抗 NMDAR1Ig G-葡萄球菌蛋白 A-胶体金复合物分子和结合标记物分子后的神经元膜进行扫描 ,三维形貌测定 ,通过颗粒度分析结果 ,明确标记物分子的特征性三维形貌 ,对比确定经过免疫胶体金结合后的 NMDA受体蛋白单分子在神经元膜表面的定位。结果显示 ,空白云母表面标记物分子为分散均匀的平均粒径为 49nm的球形颗粒 ,在神经元膜表面结合 NMDA目的受体蛋白分子后 ,免疫复合物分子呈现出粒径为 5 3 nm的散在分布球形或短棒状颗粒 ,长径约为宽径的 2倍 ,长轴截面可见典型的双峰三维结构。上述结果表明 ,NMDA受体蛋白单分子可以结合 1个或 1个以上的胶体金标记物分子 ;原子力显微镜可以在纳米尺度对神经元膜 NMDA受体蛋白进行标记和其免疫复合物的三维形貌测定。胶体金颗粒标记 ,原子力显微镜测定是免疫细胞化学新方法。

THE LOCALIZATION OF NMDA RECEPTOR PROTEIN ON CULTURE NEURONAL MEMBRANE BY ATOMIC FORCE MICROSCOPY

To discuss the application in manipulating and charging biology single molecule by atomic force microscopy and to situate the membrane protein of NMDA receptor on neuronal surface in nano scale, the anti-NMDAR1-IgG-SPA-Gold complexes molecule distributed on the surface of mica was scanned by atomic force microscopy and the characteristic 3D conformation was analysed by particle software. The 3D conformation was contrasted with the results of scanned neuronal membrane surface that combined IgG-SPA-gold complexes molecules by atomic force microscopy. Then the NMDAR protein was situated under characteristic globe of colloid gold particle. The 3D conformation of IgG-SPA-G scanned through mica surface showed that the average diameter of globe particles was 49 nm. The results of scanned through neuronal membrane combined with IgG-SPA-G showed that the immunity complex were globe or cosh shapes. Its average diameter was 53 nm and the long axis section was an typical two apexes structure. The above results demonstrate that NMDAR single molecule can combine one or two gold particles and atomic force microscopy can determine the situation and 3D conformation of NMDAR protein on neuronal membrane surface in nano scale. Colloid gold particle scanned by atomic force microscopy is a new method in immunocytochemistry.