代谢综合征药物高通量筛选模型的建立

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10 μmol L^-1 CCCP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.

Development of a high-throughput screening assay for metabolic syndrome

Mitochondria are one of the most important organelles in eukaryotic cells. In addition to supplying energy to cells, mitochondria are involved in a range of other processes, such as signaling, cellular differentiation and growth, and cell death. They have been implicated in several human diseases, including diabetes, obesity, neurodegenerative diseases and cancer. Mitochondrial membrane potential provides an index of mitochondrial functions. We developed a high-throughput screening assay for mitochondrial membrane potential in metabolic L6 myotubes. We optimized the concentration and processing time for JC-1 dye, the processing time for compounds, and the concentration of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP). The screening assay was verified with the mitochondrial inhibitors rotenone, malonate, antimycin A, oligomycin and berberine. The coefficient of variation (CV) with 10 μmol L^?1 CCCP was 5.92%, and the Z' factor was 0.575; thereby meeting the strict quality control criteria for high-throughput screening assays. Our results indicate that L6 myotubes are appropriate, sensitive and efficient for measuring mitochondrial function, and could increase the likelihood of finding novel lead compounds for the treatment of metabolic syndrome.