Influence of mutant NOTCH3 (R90C) in proliferation of oligodendrocyte lineage cells HS683 and their related molecular mechanism
10.3760/cma.j.issn.1671-8925.2016.06.006
- VernacularTitle:突变型NOTCH3(R90C)对少突胶质系细胞HS683增殖的影响及相关分子机制
- Author:
Mibo TANG
1
;
Changhe SHI
;
Bo SONG
;
Yusheng LI
;
Ting YANG
;
Chengyuan MAO
;
Yuming XU
Author Information
1. 450000郑州,郑州大学第一附属医院神经内科,河南省高等学校临床医学重点学科开放实验室
- Keywords:
Cerebral arto-somal dominant arteriopath with subcortical infarcts and leukoence-phalopathy;
NOTCH3(R90C);
Oligodendrocyte;
Cell proliferation;
p53
- From:
Chinese Journal of Neuromedicine
2016;15(6):569-574
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the influence of one kind of defective gene NOTCH3 (R90C) of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in proliferation of oligodendrocyte lineage cells HS683 and their related molecular mechanism.Methods (1) A pCMV-Sport6.0 was chosen as the expression vector and site-directed mutation was used to construct the mutant NOTCH3 (p.R90C) expression vector;eukaryotic cell transfection technique was used to respectively transfect the pCMV-Sport6.0 empty vector,wild NOTCH3 vector (p.NOTCH3) and mutant NOTCH3 (p.R90C) expression vector to HS683 cells (blank control vector group,wild NOTCH3 vector group,and mutant NOTCH3 vector group);the protein expressions of NOTCH3,p53,phosphorylated p53 and p21 were detected by Westem blotting.(2) Wild NOTCH3 vector group,mutant NOTCH3 vector group and mutant NOTCH3 vector+pifithrin-α group were divided,and after wild NOTCH3 vector (p.NOTCH3) and mutant NOTCH3 (p.R90C) vector transfection,the latter two groups were added 0 or 1 μmol/L pifithrin-α,respectively;CCK-8 assay was employed to test the proliferation oftransfected HS683 cells 24,48 and 72,and 96 h after transfection.Results (1) As compared with wild NOTCH3 vector group,mutant NOTCH3 vector group had significantly lower absorbance value 24,48 and 72 h after transfection (P＜0.05);72 h after transfection,wild NOTCH3 vector group and mutant NOTCH3 vector group had significantly higher NOTCH3 protein expression as compared with blank control vector group (P＜0.05),and mutant NOTCH3 vector group had significantly higher p53,phosphorylated-p53 and p21 protein expressions as compared with wild NOTCH3 vector group and blank control vector group (P＜0.05).(2) The absorbance value in the mutant NOTCH3 vector+pifithrin-α group was significantly increased as compared with that in the mutant NOTCH3 vector group 48,72 and 96 h after transfection (P＜0.05).Conclusion Mutation of NOTCH3 (R90C) may inhibit the proliferation of oligodendrocyte cell lineage via p53 dependent way,which might play a direct role in demyelination pathology of CADASIL caused by NOTCH3(R90C).
