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线粒体对于真核细胞至关重要,除了为细胞提供能量,还参与细胞信号转导、分化与生长、凋亡等生命过程.许多疾病的发生与线粒体功能失常密切相关,包括神经退行性疾病、糖尿病、肥胖、肿瘤等.由于线粒体膜电位是反映细胞功能状态的重要指针,因此选用了代谢性细胞L6大鼠肌管细胞建立了一种快速且高效地分析线粒体膜电位的高通量筛选模型.通过对线粒体染料JC-1浓度及孵育时间、待筛选化合物孵育时间、阳性化合物CCCP(carbonylcyanide-m-chlorophenylhydrazone)浓度等实验条件的优化,确立了筛选条件,并对鱼藤酮、丙二酸、抗霉素A、寡霉素和黄连素(berberine)等线粒体抑制剂进行验证,证实该筛选体系的可靠性.以10μmol L?1CCCP作为阳性对照,筛选体系的整体CV值(变异系数)为5.92%,Z′因子为0.575,符合高通量筛选的要求.L6肌管细胞线粒体膜电位高通量筛选模型的建立,将为发现治疗代谢综合征的新药先导化合物提供更多机会.
Mitochondria are crucial for eukaryotic cells, which not only supply energy to cells, but also participate in the life process of cell signal transduction, differentiation and growth, apoptosis, etc. Many diseases are closely related to mitochondrial dysfunction, including neurodegenerative diseases, diabetes mellitus , Obesity, cancer, etc. Since mitochondrial membrane potential is an important indicator reflecting the functional status of cells, a selection of metabolic cells in myotubes of L6 rat established a high-throughput screening model for rapid and efficient analysis of mitochondrial membrane potential. The screening conditions were established by optimizing the experimental conditions such as the concentration and time of mitochondrial dye JC-1, the incubation time of the compound to be screened, the concentration of CCCP (carbonylcyanide-m-chlorophenylhydrazone), and the screening conditions of rotenone, malonic acid, (A), oligomycin and berberine to verify the reliability of the screening system.With 10μmol L · 1CCCP as a positive control, the overall CV value of the screening system (coefficient of variation) was 5.92% Z ’factor of 0.575, in line with the requirements of high-throughput screening L6 myotube membrane mitochondrial membrane potential high-throughput screening model will be established Find a cure for drug lead compounds to provide more opportunities for the metabolic syndrome.