禹文峰， 孔　欣， 官志忠，**

(1.贵州医科大学 分子生物学重点实验室， 贵州 贵阳　550004; 2.贵州医科大学 病理学教研室， 贵州 贵阳　550004)



一种改进的Aβ1-42寡聚体的制备与鉴定方法*

禹文峰1， 孔欣2， 官志忠1，2**

(1.贵州医科大学 分子生物学重点实验室， 贵州 贵阳550004; 2.贵州医科大学 病理学教研室， 贵州 贵阳550004)

目的： 改进Aβ1-42寡聚体的制备与鉴定方式。方法： 以化学合成多肽Aβ1-42为原料在体外制备Aβ1-42寡聚体，用Western blotting进行鉴定;加入不同浓度Aβ1-42寡聚体后，用CCK-8法测定体外原代培养大鼠大脑皮质星形胶质细胞(AS)的存活率。结果： 化学合成的 Aβ1-42寡聚体分子量为4～188 kD，以64 kD最多，亦有少量4 kD单体存在；CCK-8法显示Aβ1-42寡聚体对AS有明显的神经毒性作用，并呈现剂量-效应关系。结论： 成功建立了一种改进的制备和鉴定Aβ1-42寡聚体的方法，为深入研究阿尔茨海默病提供了研究手段。

淀粉样蛋白； 阿尔茨海默病； 寡聚体； 星形胶质细胞； 免疫印迹法

[Abstract]Objective: To improve method for preparation and identification of Aβ1-42oligomer. Methods: Aβ1-42oligomer was prepared by using chemical synthetic Aβ1-42polypeptideinvitro. The prepared peptide was identified by the assay of Western blotting. In order to confirm the neurotoxicity of the peptide, primary cultured astrocytes from the cortex of rats were treated with different concentrations of Aβ1-42oligomer and the survival rates of the cells were detected via Cell Counting Kit-8(CCK-8) assay. Results: The results indicated that chemical synthetic Aβ1-42oligomer weighed from 4 to 188 kd, in which the points with 64 kd were the most. In addition, a few points with 4 kd, a monomer, was also found. Furthermore, the neurotoxicity of Aβ1-42oligomer on astroytes was significantly detected with a manner of dose-dependence. Conclusions: An improved method for preparation and identification of Aβ1-42oligomer has been successfully established, which is important in the research work of Alzheimer's disease.

[Key words]amyloid; Alzheimer disease; oligomer; astrocytes; immunoblotting

阿尔茨海默氏病(Alzheimer disease, AD)是一种常见的神经退行性疾病，AD患者表现出进行性记忆能力降低、行为和认知功能障碍。AD的主要病理改变有脑组织老年斑及神经纤维缠结形成，老年斑的主要成分是β-淀粉样蛋白(amyloid β-protein，Aβ)[1-2]。在AD的发病过程中，Aβ分子经历了从可溶态到不溶纤维状的转变。神经细胞内的Aβ生成后分泌到细胞外，累积到一定程度后就会自发聚合构成寡聚体、原纤维体及中间体，最后会形成成熟、老化的纤维状Aβ。目前认为，可溶性Aβ寡聚体是产生神经毒性作用的重要来源，比纤维状Aβ的毒性更强，在AD的发病机理中起着重要作用[3-6]。……