β-淀粉样蛋白单体及寡聚体鉴定方法的改进与优化

Modification and Optimization of Identification Methods for β-Amyloid Peptide Monomers and Oligomers

β-淀粉样蛋白(β-amyloid peptide,Aβ)经过自身聚集形成寡聚体,是导致阿尔茨海默病(Alzheimer’s disease,AD)发病的重要风险因素之一。鉴定Aβ寡聚状态对于细胞毒性和寡聚抑制的研究非常重要。本文通过优化Aβ42蛋白用量、增加交联剂、尺寸排阻色谱法(size exclusion chromatography,SEC)和硫磺素T(thioflavin T,Th T)荧光分析等方法,对Aβ42的蛋白质聚集状态进行检测。得到改进和优化的方案:(1)对电泳和电镜的优化,16.5%的Tris-tricine胶,最适合分析Aβ42寡聚状态。使用电泳和电镜对Aβ42检测时,其用量有较严格的限制。电泳的上样量应为0.5μg最佳。50μg/m L Aβ42单体浓度,最适合电镜观察;(2)对Aβ42交联的优化,终浓度为0.3mmol/L BS3,有利于对Aβ42寡聚体的区分;(3)SEC可以利用凝胶孔隙的大小,实现Aβ42单体和寡聚体在非变性条件下的分离。与Aβ40相比,Aβ42进行SEC分析时,单次上样量需达到6.75μg以上;(4)细胞毒性实验和Th T检测,可以反馈Aβ42寡聚状态的鉴定效果。上述检测方法的细化和改进,将为Aβ42肽在AD的研究中提供一定的参考。一些Aβ42特异性寡聚体的鉴定方法本文未提及,仍需要在今后的研究中进一步改进与优化。

Abstract The formation of β-amyloid peptide （AIS） oligomers is an important event in the pathogenesis of Alzheimer＇s disease （AD）. However, identification of the aggregation state of Aβ42 is essential for studies of cytotoxicity and inhibition of aggregation. A series of reasonable and simple methods were evaluated to distinguish Aβ42 monomers and oligomers based on previous studies, such as optimization of the Aβ42 amount, increase of the cross-linker, size exclusion chromatography （SEC） and thioflavin T （ThT） fluorescence analysis, etc. The following modification and optimization results are obtained： （1） Optimization of electrophoresis and electron microscopy, which are commonly usect tor aetecuon of Aβ42 monomers and oligomers, has strict requirements for the amount of Aβ42. A concentration of 16.5% tris- tricine gel is best suited for electrophoretic analysis of Aβ42. For silver staining, theAβ42 quantity in each lane is 0.5 μg. Aβ42 monomer at a concentration of 50μg/mL is most suitable for electron microscope observation. （2） Addition of crosslinker BS3 at the final concentration of 0.3 mmol/L was found to be more effective for differentiation of Aβ42 aggregation forms. （3） Size exclusion chromatography （SEC） can be used to separateAβ42 monomers and oligomers depending on pore size under non-denaturing conditions, but previous reports did not show the amount of Aβ42. Our methods provided detailed information for the amounts of Aβ42. The amount of Aβ42 sample added must be higher than 6.75μg per time in SEC analysis when compared with Aβ40. （4） Cytotoxicity test and ThT analysis can be used to reveal whether a method is suitable for identifying the Aβ42 oligomerization state. Refinement and improvement of these methods will provide a reference for the study of Aβ42 in patients with Alzheimer＇ s disease. Some methods for the identification of specific Aβ42 oligomers have not been mentioned in this paper, and further studies are needed.