β-淀粉样肽寡聚体的制备及鉴定

Preparation and identification of oligomeric amyloid-β peptide

[目的]制备β-淀粉样肽(amyloid-βpeptide,Aβ)的可溶性寡聚体。[方法]选取化学合成的Aβ25-35、Aβ1-42及对照肽Aβ35-25,体外37℃孵育7 d制备Aβ寡聚体。采用dot blotting,免疫印迹鉴定Aβ的聚集状态;并通过TUNEL法检测Aβ的神经毒性。[结果]经dot blotting和免疫印迹检测,显示本研究制备获得的Aβ25-35和Aβ1-42均为寡聚体,且Aβ25-35寡聚体的分子量以2 kDa、3 kDa和17 kDa为主,Aβ1-42分子量主要为4kDa和8kDa,而对照肽Aβ35-25并不能形成寡聚体。TUNEL法则显示Aβ25-35寡聚体可以引起SH-SY5Y细胞凋亡。[结论]在体外成功建立制备Aβ寡聚体的方法,且通过形态学实验证实该Aβ有神经毒性,为后续研究阿尔兹海默症的致病机制奠定实验基础。

[ Objective ] Preparation of soluble oligomeric amyloid -β peptide （Aβ）. [ Methods ] Chemically synthetic Aβ25 - 35 ,Aβ1 -42 or Aβ35 -25 （control peptide of Aβ25 -35 ） was respectively prepared through 37℃ incubation for 7 days in vitro. The oligomeric form of Aβ was observed and assessed via dot blotting and Western blot. Furthermore,TUNEL staining was used to verify the neurotoxicity of Aβ25 -35 oligomer. [ Results] The dot blotting and Western blot results showed that Aβ25 - 35 and Aβ1 -42 were both oligomers,while Aβ35 -25 was not. Aβ25 -35 oligomers were appeared as a ladder in the molecu- lar weight about 2 kDa,3 kDa and 17 kDa. And Aβ1 -42 oligomers were mainly at 4kDa and 8kDa. In addition,it was shown that Aβ25 -35 oligomer induced SH -SYSY cells apoptosis by TUNEL staining. [ Conclusion] The research successfully established a method of A[3 oligomers preparation in vitro and the Aβ was proved to be neurotoxicity by morphological experiment, which will lay the experimental foundation of pathogenic mechanism of Alzheimer＇ s disease.