Preparation and identification of amyloid-β protein 1-42 oligomers and their effect on astrocytes
10.3760/cma.j.issn.1671-8925.2017.02.002
- VernacularTitle:β-淀粉样蛋白1-42寡聚体的制备、鉴定及其对星形胶质细胞的作用
- Author:
Xiaoyu HUANG
1
;
Canhong YANG
;
Xiaomin HUANG
;
Hantao MAI
;
Tianming LYU
Author Information
1. 南方医科大学第三附属医院神经内科
- Keywords:
Alzheimer's disease;
Amyloid-β protein;
Astrocyte;
Aquaporin-4
- From:
Chinese Journal of Neuromedicine
2017;16(2):114-120
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the preparation and identification of amyloid-β protein 1-42 (Aβ1-42) oligomers and their effect on in vitro cultured astrocytes in mice.Methods (1) Aβ1-42peptides were dissolved and 100 μ mol/L Aβ1-42 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 Aβ-42 was observed under electron microscope and the degrees of Aβ1-42 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 ofAβ1-42 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 Aβ1-42oligomers 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 Aβ1-42 oligomer treatment groups had activated astrocytes:enlarged soma,increased cell processes and increased GFAP fluorescence intensity were noted;Western blotting indicated that following the increased oligomer concentrations,the protein expressions of GFAP and AQP4 increased:as compared with the 0 μmol/L Aβ1-42 oligomer treatment group,the 10 and 50 μmol/L Aβ1-42 oligomer treatment groups had significantly increased GFAP protein expression (P＜0.05);and as compared with the 0 μmol/L Aβ1-42 oligomer treatment group,the one,10,and 50 μmol/L Aβ1-42 oligomer treatment groups had significantly increased AQP4 protein expression (P＜0.05) Conclusions The Aβ1-42 oligomers could be prepared with 100 μmol/L peptide under 4 ℃ for 24 h.Aβ1-42 oligomers could activate astrocytes and up-regulate the AQP4 expression,which might be a self protective mechanism.
