Skin fibroblasts derived from Leber's hereditary optic neuropathy patients present mitochondrial dysfunction
10.3760/cma.j.cn511434-20220627-00385
- VernacularTitle:Leber遗传性视神经病变患者永生化皮肤成纤维细胞线粒体功能探讨
- Author:
Shun YAO
1
;
Mingzhu YANG
;
Ya LI
;
Bo LEI
Author Information
1. 河南省人民医院 河南省立眼科医院, 郑州 450003
- Keywords:
Optic atrophy, hereditary, Leber;
Fibroblasts;
Skin;
Mitochondria;
Cell survival
- From:
Chinese Journal of Ocular Fundus Diseases
2022;38(8):675-680
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the feasibility of immoribund skin fibroblast cell line derived from Leber's hereditary optic neuropathy (LHON) patients as a cell model.Methods:A basic research. Two LHON patients and 2 healthy volunteers were recruited from Department of Ophthalmology of Genetic Clinic of Henan Provincial Eye Hospital. The skin tissue of participants was obtained, and the 4 immortalized skin fibroblasts were constructed by SV40 virus infection, including 2 LHON patient cells (LHON-1 and LHON-2 cells) and 2 healthy volunteers cells (NC-1 and NC-2 cells). Mitochondrial morphology in cells was observed by electron microscope. The levels of reactive oxygen species (ROS), nicotinamide adenine dinucleotide-oxidation state (NAD +), nicotinamide adenine dinucleotide-reduction state (NADH) and adenosine triphosphate (ATP) in fibroblasts were detected. Cellular oxygen consumption was measured by seahorse mitochondrial pressure assay. Cell viability was detected using cell counting kit-8 (CCK8). One-way ANOVA was performed to compare the levels of ROS, NAD +, NADH and ATP in LHON and NC cells, as well as basal oxygen consumption, maximal oxygen consumption, ATP-coupled oxygen consumption, and cell viability. Results:Compared with NC-1 and NC-2, the number of mitochondrial crest in LHON fibroblasts was significantly reduced, indicating abnormal mitochondrial morphology. Biochemical analysis showed that ROS levels in LHON cells increased, but NAD +/NADH and ATP levels decreased, and the oxygen consumption was significantly inhibited, indicating the presence of mitochondrial damage and respiratory dysfunction. The results of CCK-8 detection showed that the survival ability of LHON-1 and LHON-2 cells was worse under stress conditions. Conclusion:Immortalized skin fibroblast cell lines from LHON patients presented mitochondrial dysfunction.
