摘要
目的:利用原子力显微镜(AFM)观察β-淀粉样蛋白(Aβ_(1-42))在不同聚集状态下的形态结构。方法:制备寡聚体:用六氟异丙醇溶解Aβ_(1-42)单体,通风干燥后重悬于二甲基亚砜,以PBS稀释后4℃孵育24 h;按常规方法制备Aβ单体和纤维体;用AFM的轻敲模式及相位成像技术观察不同聚集状态Aβ的形态结构,并与单体和纤维体进行比较。结果:在制备寡聚体样本中,可观察到不同形状的Aβ_(1-42)聚集体,包括球状寡聚体、环状结构、初级原纤维、成熟原纤维体等。初级原纤维呈串珠样链状结构,成熟原纤维呈致密的绳状结构。寡聚体高度为2~6nm,均值为4.39 nm;原纤维的长度约1μm,高度为1.5 nm^4 nm。单体分布均匀,呈颗粒状,平均高度为0.65±0.16 nm。纤维长度为2μm以上,高度为2~4 nm。结论:1用AFM可观察到Aβ单体、纤维和不同聚集状态的中间体,AFM技术是研究蛋白质聚集过程中动态变化的较好工具;2Aβ经PBS处理,可孵育出多种Aβ_(1-42)中间体:球状体、环状结构和原纤维;3本实验将为研究阿尔茨海默病(AD)的发病机制和开发治疗AD新疗法提供实验依据。
Objective： To study the different aggregation morphology of β-amyloid （Aβ1-42） using atomic force microscope （AFM）. Methods： Preparation of Aβ Olgomers： Aβ142 peptide was dissolved in cold hexafluoro-2-propanol （HFIP）. After evaporation, the resulting film was dissolved in anhydrous DMSO and diluted into PBS aged 24 h at 4℃, and prepare Aβ monomers and fibrils by conventional methods. The tapping mode and phase imaging technology of AFM were applied to observe the different aggregation morphology of Aβ1-42 and compare with monomers and fibrils. Results： AFM images contain a spectrum of Aβ structures, including globular oligomers with diameters varying from 2 to 6 nm （ mean 4.39 nm）, ring-like structures, protofibrillar structures with lengths less than 1 μm and heights ranging from 1.5 to 4 nm. Primary protofibrils appear to be beaded chain structures, and ripe protofibrils like tight rope-like structures. Granula monomers evenly distributed with an average height of 0.65 ± 0.16 nm. The length of fibrils are longer than 2 μm and the height ranged from 2 - 4 nm. Conclusions： The different aggregation species of Aβ142, such as monomers, fibrils and different state of aggregation intermediates, could be observed using AFM, which is a good tool to study the dynamic changes in the process of protein aggregation; After incubated in PBS, Aβ could express a variety of Aβ1-42 peptide intermediates, including spheroids, ring-like structures and fibrils. The present study will provide experimental evidence for the reasearch of the pathogenesis of AD and the development of new treatments for AD.
出处
《神经解剖学杂志》
CAS
CSCD
北大核心
2016年第1期77-81,共5页
Chinese Journal of Neuroanatomy
基金
国家自然科学基金(81260174)
