Liu Qian, Jiang Zhaoyan, Gu Aihua
State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University; Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University; Center of Gallbladder Disease, Shanghai East Hospital, Institute of Gallstone Disease, Tongji University School of Medicine.
Center of Gallbladder Disease, Shanghai East Hospital, Institute of Gallstone Disease, Tongji University School of Medicine;
J Vis Exp. 2020 Sep 16(163). doi: 10.3791/56800.
This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to β-hexachlorocyclohexane (β-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with β-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.
本文介绍了检测肝脏线粒体功能的详细方法，以便更好地理解环境有机氯农药（OCPs）导致肝细胞代谢紊乱的原因。将HepG2细胞以一般人群体内暴露的等效剂量暴露于β-六氯环己烷（β-HCH）24小时。通过透射电子显微镜（TEM）检查肝细胞超微结构以显示线粒体损伤。通过与β-HCH孵育的HepG2细胞中的线粒体荧光强度、三磷酸腺苷（ATP）水平、耗氧率（OCR）和线粒体膜电位（MMP）进一步评估线粒体功能。用线粒体绿色荧光探针染色后的线粒体荧光强度用荧光显微镜观察。采用荧光素-荧光素酶反应测定ATP水平。用阳离子染料JC-1检测MMP并在流式细胞仪下分析。用细胞外通量分析仪测量OCR。总之，这些方案用于检测肝细胞中的线粒体功能以研究线粒体损伤。
