神经元膜NMDA受体蛋白免疫复合物单分子纳米结构及定位AFM比较研究

目的探讨NMDAr(N-甲基-D-天冬氨酸受体)在神经细胞膜表面定位，对比不同成像方法的特点，建立纳米尺度神经细胞膜表面蛋白单分子三维标记的免疫细胞化学新方法。方法应用原子力显微镜，对分布在云母表面的抗NMDAR1亚单位IgG-葡萄球菌蛋白A-胶体金颗粒免疫标记复合物分子进行扫描，明确其特定的三维结构作为对照标准，然后扫描结合了免疫标记复合物分子的神经细胞膜表面，对表面形貌作出对比后确定目的抗原的定位和复合物三维结构。并与光镜、激光共聚焦、扫描电镜等方法进行对比。结果在空白云母表面，免疫复合物分子在原子力显微镜下呈现出均匀分散粒径为49nm的特征性球形结构。在神经元表面结合免疫标记物后可以发现有大量的粒径为53nm的球形或双球形(短棒状)结构，颗粒均匀，截面为双峰或单峰。光镜下染色成片状结果，在共聚焦显微镜下荧光呈颗粒点状分布，扫描电镜结果为单个或结合颗粒，缺乏三维成像能力。结论原子力显微镜下免疫胶体金标记技术可以在纳米尺度对目的膜受体蛋白进行定位和表面三维结构测定。NM-DA受体蛋白可以结合一个或两个胶体金复合物，与传统成像手段相比，原子力显微镜下胶体金标记方法可以对单个膜NMDA受体蛋白抗原进行定位、定性、定量标记测定。

Comparative study of the localization and conformation of immunocomplexes single molecule of NMDA receptor protein on neuron membrane by AFM

Objective In order to study the localization of NMDA receptor protein on the neuron membrane, a new immunocytochemistry method to visualize and quantify 3D earmark the membrane protein in nanometer scale has been achieved by atomic force microscope (AFM) with labeled colloid gold particle. Methods First step, the distribution of the anti-NMDAR1 IgG-SPA-gold complexes molecule on the surface of mica had been scanned by AFM and the characteristic 3D conformation was analyzed by particle software. Then the 3D conformation was contrasted with the IgG-SPA-gold complexes molecules combined with the neuron membrane surface and that were compared with the results of light microscope, laser confocal microscope and scanning electron microscope. Results The 3D conformation of IgG-SPA-gold scanned through mica surface shows the average diameter of globe particles is 49 nm .The neuron membrane NMDA receptor protein combined with IgG-SPA-gold immunocomplexes are globe or cosh shapes .Its average diameter is 53 nm. The long axis section is a typical two apexes structure. Conclusion AFM could determine the situation and 3D conformation of NMDAR protein on neuron membrane surface in nanometer scale. NMDAR single molecule could combine with one or two colloidal gold particles. Colloidal gold particle scanned by AFM in situ is a new detecting method of immunocytochemistry.