Construction of mutant NDUFS7 plasmid and its effects on neural cells

SHEN Jie; ZHA Qian; GAO Xiangdong; CHEN Song

doi:10.11665/j.issn.1000-5048.20200512

Journal of China Pharmaceutical University > 2020 > 51(5): 599-606. > DOI: 10.11665/j.issn.1000-5048.20200512

SHEN Jie, ZHA Qian, GAO Xiangdong, CHEN Song. Construction of mutant NDUFS7 plasmid and its effects on neural cells[J]. Journal of China Pharmaceutical University, 2020, 51(5): 599-606. DOI: 10.11665/j.issn.1000-5048.20200512

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Construction of mutant NDUFS7 plasmid and its effects on neural cells

Funds: This study was supported by the National Natural Science Foundation of China (No.81673435, No.81872850)

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Received Date: April 03, 2020
Revised Date: June 28, 2020

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Abstract

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.
- NDUFS7,
- dopaminergic neurons,
- cell apoptosis,
- mitochondrial membrane potential

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