基于负染-电镜的A体外聚集表征测试要点分析
张 钊，董润敏，张 莹*，孙维敏，王 荷，王 欢，杨海强，温 杰基于负染-电镜的Ab体外聚集表征测试要点分析
张 钊，董润敏，张 莹*，孙维敏，王 荷，王 欢，杨海强，温 杰
（北京交通大学理学院生命科学与生物工程研究院电镜实验室，北京100044）
摘 要 本文应用电镜负染色技术，对阿尔茨海默病（Alzheimer’s disease, AD）中所涉及的b淀粉样肽（b-amyloid peptide，Ab）的聚集规律进行了分析。通过对样品的浓度、pH值、温度和时间等设置系列梯度，以及调整负染试剂的种类、浓度和染色时间，优化了Ab寡聚体样品制备以及电镜观察的最佳条件，获得了清晰的Aβ纤维图像并用以估算纤维长度。实验结果表明：经过六氟异丙醇给予单体化处理，并溶于无水二甲基亚砜（DMSO）的Ab（100 mmol·L-1），在pH7.2的PBS中4℃孵育24 h，Ab寡聚体形态清晰，且Aβ42寡聚体比Aβ40寡聚体聚集趋势更明显。在pH 8.0的硼酸缓冲液中，Ab42（40 mmol·L-1）在37℃孵育72 h后，纤维形成明显；而Ab纤维样品经过煮沸后再制备负染样品，所得电镜图像更为清晰，便于对纤维长度和结构进一步分析。因此电镜负染色技术，可作为一种快捷，直观的Ab体外聚集形貌表征的质控方法。
关键词 透射电子显微镜；b-淀粉样蛋白；聚集；负染色技术
中图分类号：Q71；Q336 文献标识码：A doi:10.3969/j.issn.1000-6281.2016.05.008
Essentials analysis on negative staining-based TEM morphological characterization of Aβ aggregation in vitro
ZHANG Zhao，DONG Run-min，ZHANG Ying *，SUN Wei-min，WANG He，WANG Huan，YANG Hai-qiang，WEN Jie
（Electron Microscopy Lab, Institute of Life Sciences and Bioengineering, School of Science, Beijing Jiaotong University,Beijing 100044，China）
Abstract In this study, the negative staining electron microscopywas used to analyze the accumulation regularity of β- amyloid (Aβ) in Alzheimer’s disease (AD). The parameters including Aβ concentration, pH of reaction buffer, temperature and incubation time were adjusted to form Aβ oligomers and fibrils, and different negative staining reagents as well as staining times were also tried in order to obtain the best TEM images for Aβ assembly. At the same time, the images of Aβ fibrils were improved to analyze the length of Aβ fibrils. The results showed that the classical Aβ oligomers were detected after the proper preparation in which the Aβ peptides (100 mmol·L-1) were treated with HFIP, DMSO and incubated in phosphate buffer solution (pH7.2) 4℃for 24 h. The oligomers of Aβ42 assembled much more obviously than Aβ40. In addition, the Ab (40 mmol·L-1) fibrils from 72 h-incubation (37℃) within boric acid buffer (pH 8.0) were clear under TEM. Furthermore, the negative staining images would become more distinguishable after the fibrils samples were boiled in water. Therefore, TEM is an easy-handle, convenient, fast and direct method for the investigation of the morphology of Aβ aggregation in vitro.
Keywords transmission electron microscopy (TEM)；β-amyloid protein (Aβ)；aggregation；negative staining