Construction of mutant NDUFS7 plasmid and its effects on neural cells
10.11665/j.issn.1000-5048.20200512
- VernacularTitle:突变型NDUFS7质粒的构建及其对神经细胞的影响
- Author:
Jie SHEN
1
;
Qian ZHA
;
Xiangdong GAO
;
Song CHEN
Author Information
1. 中国药科大学生命科学与技术学院
- Publication Type:Journal Article
- Keywords:
NDUFS7;
dopaminergic neurons;
cell apoptosis;
mitochondrial membrane potential
- From:
Journal of China Pharmaceutical University
2020;51(5):599-606
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the effects of NDUFS7 gene mutation on neurons, the mutant plasmid of pcDNA3.1(+)-NDUFS7 Q208STOP was constructed and transfected into differentiated SH-SY5Y cells. The effect of transfecting mutant plasmid on the viability of dopaminergic neural cells was detected by MTT assay. The effect of transfection of mutant plasmid on apoptosis was detected by Annexin Ⅴ-FITC/PI staining followed by flow cytometry assay. The changes in the expression levels of apoptosis-related proteins Bax and Bcl-2 in cells after transfection of mutant plasmid were detected by Western blot. The effects of transfection of mutant plasmid on the mitochondrial membrane potential in differentiated SH-SY5Y cells and the intervention effect of antioxidant Trolox were examined using JC-1 fluorescent probe. The intervention effect of Trolox on the apoptosis of differentiated SH-SY5Y cells transfected with mutant plasmid was detected by PI/Hoechst staining. The results showed that the subunit mutation of mitochondrial complex I in dopaminergic neurons could lead to decreased neuronal viability and increased apoptosis, while antioxidants could alleviate the abnormal mitochondrial membrane potential and apoptosis caused by transfection of mutant plasmids, suggesting that transfection of mutant plasmid of NDUFS7 gene could lead to apoptosis by causing abnormal mitochondrial function in dopaminergic neurons.
