突变型NDUFS7质粒的构建及其对神经细胞的影响

沈杰; 查倩; 高向东; 陈松

doi:10.11665/j.issn.1000-5048.20200512

突变型NDUFS7质粒的构建及其对神经细胞的影响

中国药科大学生命科学与技术学院江苏省生物药物成药性研究重点实验室，南京 211198

基金项目: 国家自然科学基金资助项目(No.81673435,No.81872850)

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出版历程
- 收稿日期: 2020-04-03
- 修回日期: 2020-06-28
- 刊出日期: 2020-10-24

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)

摘要

摘要: 为了研究NDUFS7基因突变对神经元的影响，通过构建pcDNA3.1（+）-NDUFS7 Q208STOP突变质粒，并将其转入分化后SH-SY5Y细胞。采用MTT法检测转染突变质粒对多巴胺能神经细胞活力的影响；Annexin Ⅴ-FITC/PI双染结合流式细胞术检测转染突变质粒对细胞凋亡情况的影响；Western blot法检测转染突变质粒后细胞内凋亡相关蛋白Bax、Bcl-2表达水平的变化；采用JC-1探针检测转染突变质粒对分化后SH-SY5Y细胞内线粒体膜电位的影响及抗氧化剂Trolox的干预作用；最后PI/Hoechst双染检测Trolox对转染突变质粒的分化后SH-SY5Y细胞凋亡情况的干预作用。结果显示，多巴胺能神经细胞内线粒体复合体Ⅰ亚基突变能够导致神经细胞活力下降，凋亡增多，而抗氧化剂能够缓解转染突变质粒导致的线粒体膜电位异常和细胞凋亡，提示转染NDUFS7基因突变质粒能通过引起多巴胺能神经细胞内线粒体功能异常从而导致细胞凋亡。
- NDUFS7 /
- 多巴胺能神经元 /
- 细胞凋亡 /
- 线粒体膜电位
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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文章访问数: 233
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突变型NDUFS7质粒的构建及其对神经细胞的影响

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出版历程

Construction of mutant NDUFS7 plasmid and its effects on neural cells

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出版历程

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