原子力显微镜对自由基损伤的红细胞膜表面精细结构的研究

Study of Fine Structures of the Natural Surface of Free Radical Treated Red Blood Cells with Atomic Force Microscopy

目的直接观察自由基损伤、加女贞子保护、Ｈ２Ｏ２作用等因素对红细胞膜分子形貌学的影响。方法利用０．１纳米分辨率水平的原子力显微镜对单纯戊二醛固定的经由自由基、Ｈ２Ｏ２和女贞子作用的红细胞膜表面进行了纳米尺度的扫描观察，并与扫描电子显微镜的结果进行比较。结果自由基作用后的红细胞膜表面蛋白颗粒呈现直径从几十～几百纳米的、大小不等的团块样聚集体和膜表面纳米水平的凹凸改变，恢复３小时后红细胞的上述变化减轻，加女贞子保护或Ｈ２Ｏ２作用只引起红细胞膜表面蛋白颗粒轻度聚集和蛋白颗粒间缝隙增加。结论（１）原子力显微镜具有制样简单、无须染色和临界点干燥、可在室温和空气环境中直接观察活的或固定的生物样本的分子形貌学等优点；（２）原子力显微镜可清晰分辨自由基、Ｈ２Ｏ２、女贞子作用后细胞膜表面在分子水平的形貌学变化特征，即Ｈ２Ｏ２和女贞子作用后的红细胞膜表面分子形貌学变化程度轻于自由基的作用；（３）原子力显微镜可对所观察到的红细胞膜表面分子形貌学改变进行三维定量分析。

Objective\ To observe the effects of free radicals(Fr),ligustrum lucidum ait (LLA)+Fr,and H 2O 2 on the natural molecular morphology of red blood cell membrane surface directly.Methods\ Using an atomic force microscopy(AFM) with 0 1 nanometer resolution to scann the glutaraldehyde fixed membrane surface of RBCs,which have been treated by Fr,LLA+Fr,H 2O 2 respectively;and on nanometer range,in room temperature and air conditions;and to compare with SEM images results of.Results\ After Fr treatment, visual polymers with various sizes,ranging from tens to hundred nanometers,aopear at protein particles of RBC membrane surface the membrane surface roughness being at nanometer level.After recovering for 3h,these particle changes decreased partly.LLA+Fr or H 2O 2 treatment just resulted in smaller size polymers formation,and increase of seams as well between particles.Conclusion\ (1)AFM offers relatively quick and easy specimen preparations than SEM,without requirements to dehydrate in graded ethanol and dry at critical point,and no sputter coating.It also offers direct observing living or fixed biological specimen's surface to get molecular topography at room temperature and air conditions;(2)AFM can clearly distinguish the molecular characteristic structures of RBC membrane surface after treatment with Fr,LLA+Fr or H 2O 2.That is,the surface change of the RBC membrane,treated by LLA+Fr and H 2O 2,were less then changes those,treated with Fr at molecular topographical level;(3)AFM can do 3 dimensional quantitatie analysis for the observed RBC molecular morphology at membrane surface.