摘要
目的探讨伴皮质下梗死和白质脑病的常染色体显性遗传性脑动脉病（CADASIL）的一种突变型基因NOTCH3（R90C）对少突胶质系细胞HS683增殖能力的影响及相关的分子机制。方法（1）以pCMV—Spot6．0为表达载体，采用定点突变法构建突变型基因NOTCH3（R90C）表达载体，真核细胞转染技术分别转染pCMV-Spot6．0空白对照载体、野生型NOTCH3（p．NOTCH3）质粒与突变型NOTCH3（p．R90C）质粒到HS683细胞，分别为转染空白对照载体组、转染野生型NOTCH3基因组、转染突变型NOTCH3基因组。转染后24h、48h、72h、96h用CCK8法检测3组细胞的增殖水平；转染后72hWestemblotting检测细胞NOTCH3蛋白和细胞周期相关蛋白p53，磷酸化（p）．p53与p21的表达。（2）实验分转染野生型NOTCH3基因组、转染突变型NOTCH3基因组、转染突变型NOTCH3基因＋Pifithrin-α组，分别转染野生型、突变型、突变型NOTCH3基因，后2组再分别加入0μmol／L、1μmol／L的Pifithrin-α。转染后24h、48h、72h、96h采用CCK．8法检测细胞的增殖水平。结果（1）转染48h、72h、96h后，与转染野生型NOTCH3基因组比较，转染突变型NOTCH3基因组的细胞吸光度值降低，差异有统计学意义（P〈0．05）；转染72h后，转染野生型NOTCH3基因组与转染突变型NOTCH3基因组细胞的NOTCH3蛋白表达量显著高于转染空白对照载体组：Western blotting检测显示转染突变型NOTCH3基因组细胞增殖周期相关蛋白p53、P-p53以及p21的表达量均明显高于转染野生型NOTCH3基因组和转染空白对照载体组，差异有统计学意义（P〈0．05）；（2）转染后48h、72h、96h，与转染突变型NOTCH3基因组比较，转染突变型NOTCH3基因＋Pifithrin—α组的细胞吸光度值增加，差异有统计学意义（P〈0．05）。结论突变型NOTCH3（R90C）蛋白通过p53依赖的方式抑制少突胶质细胞的增殖．这可能是NOTCH3（R90C）突变导致CADAS
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 Western 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）.
出处
《中华神经医学杂志》
CAS
CSCD
北大核心
2016年第6期569-574,共6页
Chinese Journal of Neuromedicine
基金
国家自然科学基金（81530037、U1404311）
