NMDA损伤及MK-801保护作用后原代培养神经元膜AFM形貌对比研究

Primary culture neuron membrane topography after injury by NMDA and protection by MK-801 under AFM

目的 对比观察正常培养、NMDA损伤及MK-801保护皮层神经元膜表面三维构象形貌改变。方法 分别应用光镜、扫描电镜和分辨率为0.1～0.01nm的原子力显微镜(atomic force microscope,AFM)对戊二醛固定的正常原代培养大鼠皮层神经细胞膜,NMDA损伤及MK-801保护后神经元膜表面进行纳米尺度的扫描观测,结果进行比较。结果 正常神经元膜表面光滑,起伏均匀,隆起的颗粒状蛋白密集,间隔规律。NMDA损伤后神经元破碎,崩解,膜失去连续性,颗粒蛋白质聚集成大的隆起。脂质凹陷加深,间距增宽,可见空穴状凹陷。MK-801保护作用神经元膜皱折增加,边缘粗糙,起伏程度介于前两者之间。结论 (1)AFM分辨率高,制样简单,可对活的或固定的生物样品在自然条件下进行直接观测。(2)AFM较SEM能更细微地分辩损伤保护作用后引起的细胞膜表面三维形貌改变并可以做出定量分析。(3)NMDA作用后膜结构开始解体,膜蛋白颗粒聚集增大,脂质凹陷加深,间距增宽,表面粗糙度增加。(4)对于AFM应用于生物样品的检测,还应该在制样,提高观测分辩率,增加标记物等方面做进一步研究。

Objective To comparatively observe the changes of three-dimension topography in culture neuron membrane respectively by NMDA injury, MK-801 protection and normal cultivation. Methods We observed and measured the rat dermal epithelium neuron membrane fixed by glutaraldehyhyde and then cultured primarily, injured with NMDA and protected by MK-801 respectively under light microscope (LM), scanning electric microscope (SEM) and atomic force microscope (AFM) with resolution of 0.01-0.1 nm. Results The surface of normal neuron membrane is smoother and more equitable and its dense projected granule proteins are of regular granularity. The neuron injured by NMDA broke, its membrane out of continuity and granule proteins gathering with prominence, lipid incisure deeper with widened spacing. The neuron membrane shielded by MK -801 had increased in creases and the roughness of margin fell between the preceding two groups. Conclusion (1) The high resolution of AFM makes the experimental model easy. (2) AFM is better in distinguishing the three-dimension surfaces in neuron membrane after injury and protection than SEM. (3 )The membrane injured by NMDA b ecomes disintegrated, its protein particles gathering augmenting, its lipid incisure deeper, the spacing widening and the surface rougher. (4)AFM ifused in the bio-test needs further study in sampling, increase of resolution, and addition of markers.