摘要
目的探讨β-淀粉样蛋白1-42（Aβ1-42）寡聚体的制备和鉴定方法，并探讨其对体外培养的小鼠星形胶质细胞的作用。方法（1）溶解Aβ1-42越多肽后取100μmol／LAβ1-42多肽母液，分别经4℃孵育24h、4℃孵育72h、37℃孵育24h、37℃孵育72h处理，应用电子显微镜观察和Westem blotting对不同孵育条件制备的Aβ1-42进行鉴定。（2）将不同浓度的A13lm寡聚体（由Aβ1-42多肽母液经4℃孵育24h制备1加人到体外培养的星形胶质细胞中，采用CCK-8法检测不同浓度Aβ1-42寡聚体（0、0．1、0．5、1、5、10、50、100μmol／L）处理后星形胶质细胞存活率的变化，采用免疫荧光染色检测不同浓度Aβ1-42寡聚体（0、1、10、50μmol／L）处理后星形胶质细胞的形态变化，采用Western blotting检测不同浓度Aβ1-42寡聚体（0、1、10、50Aβ1-42mol／L）处理后胶质纤维酸性蛋白（GFAP）及水通道蛋白-4（AQP4）表达的改变。结果（1）Aβ1-42多肽经不同孵育条件处理后，在电子显微镜观察和Western blotting检测中分别表现出不同形态和聚合度，其中经4℃孵育24h制备的Aβ1-42寡聚体主要呈10nm颗粒状和短丝状物质的混合体；相对分子质量10000左右的蛋白表达量减少，相对分子质量15000～25000的蛋白表达量增多。（2）不同浓度Aβ1-42寡聚体刺激星形胶质细胞24h后，CCK-8法检测结果显示，细胞存活率随着浓度增加逐渐升高，其中与0 μmol／L组比较，10、50及100μmol／LAβ1-42寡聚体组细胞存活率显著增加，差异均有统计学意义衅0．05）；免疫荧光染色观察发现，与0μmol／L组比较，1、10及50μmol／LAβ1-42寡聚体组星形胶质细胞呈活化状态，表现为胞体增大，细胞突起增多、伸长，呈放射状，GFAP荧光强度增强；Westem blotting检测结果显示，随着Aβ1-42寡聚体浓度的增加，GFAP及AQP4蛋白表达均有升高趋势，其中与0 μmol／L组比较，10及5
Objective To explore the preparation and identification of amyloid-[3 protein 1-42 （Aβ1-42） oligomers and their effect on in vitro cultured astrocytes in mice. Methods （1） Aβ1-42peptides were dissolved and 100 μmol/L AI3tm polypeptide was chosen as mother liquor; and then, they were incubated under different conditions （4 ℃ for 24 h, 4 ℃ for 72 h, 37 ℃ for 24 h and 37 ℃ for 72 h）; the form of Aβ1-42 was observed under electron microscope and the degrees of AI31.42 polymerization were detected by Western blotting. （2） Aβ1-42 oligomers of different concentrations （Aβ1-42 polypeptide as mother liquor being incubated under condition of 4 ℃ for 24 h） were added into the in vitro cultured astrocytes; CCK-8 assay was used to detect the effects of Aβ1-42 oligomers （0, 0.1, 0.5, 1, 5, 10, 50, and 100 μmol/L） on astrocytic viability; after 0, 1, 10, and 50 μmol/L Aβ1-42 oligomers treatment, immunofluorescence was employed to detect the morphological changes of astrocytes and Western blotting was used to detect the glial fibrillary acidic protein （GFAP） and aquaporin-4 （AQP4） expressions. Results （1） Different forms and degrees of polymerization of Aβ1-42 could be observed by electron microscope and Western blotting： 100 μmol/L Aβ1-42 polypeptides could induce 10 nm granulated mixture of Aβ1-42 oligomers at 4 ℃ incubation for 24 h; proteins with relative molecular mass of 10 000 had decreased expression, and those of 15 000-25 000 had increased expression. （2） Twenty-four h after Aβ1-42 oligomers treatment, the viability of astrocytes was increased gradually： as compared with the 0 μmol/L Aβ1-42 oligomer treatment group, the 10, 50 and 100 μmol/L Aβ1-42 oligomer treatment groups had significantly increased viability of astrocytes （P〈0.05）; immunofluorescent staining indicated that as compared with the 0 μmol/L Aβ1-42 oligomer treatment group, the one, 10, and 50 μmol/L A131.42 oligomer treatment groups had activated astrocytes： enlarged so
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2017年第2期114-120,共7页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（61072033）
广东省自然科学基金（8151051501000053、2014A030313273）
