Zahrebelski G, Nieminen A L, al-Ghoul K, Qian T, Herman B, Lemasters J J
Department of Cell Biology & Anatomy, School of Medicine, University of North Carolina at Chapel Hill 27599-7090, USA.
Hepatology. 1995 May;21(5):1361-72.
The aim of this study was to evaluate changes in the subcellular organelles of cultured hepatocytes by laser scanning confocal microscopy during chemical hypoxia with cyanide and iodoacetate, inhibitors of mitochondrial respiration and glycolysis, respectively. Parameter-specific fluorophores used were calcein for cell topography and membrane permeability, rhodamine-dextran for lysosomes, rhodamine 123 and tetramethylrhodamine methylester (TMRM) for mitochondrial membrane potential (delta psi) and propidium iodide for loss of cell viability. During the first 30 to 40 minutes of chemical hypoxia to cultured hepatocytes, numerous surface blebs formed and cell volume increased, but delta psi decreased relatively little. Subsequently, the nonspecific permeability of mitochondrial membranes increased, and mitochondria depolarized. These events were followed a few minutes later by disintegration of individual lysosomes. After a few more minutes, viability was lost as indicated by bleb rupture, gross plasma membrane permeability to calcein, and nuclear labeling with propidium iodide. Thus, the following sequence of intracellular events occurred during chemical hypoxia: adenosine triphosphate (ATP) depletion, bleb formation with cellular swelling, onset of a mitochondrial permeability transition, disintegration of lysosomes, plasma membrane failure from bleb rupture, and cell death. Any explanation of the pathophysiology of hypoxic injury must take into account this unique sequence of events.
本研究的目的是通过激光扫描共聚焦显微镜评估在化学性缺氧过程中培养的肝细胞亚细胞器的变化，化学性缺氧分别由线粒体呼吸抑制剂氰化物和糖酵解抑制剂碘乙酸盐引发。所使用的参数特异性荧光团如下：钙黄绿素用于观察细胞形态和膜通透性，罗丹明 - 葡聚糖用于观察溶酶体，罗丹明123和四甲基罗丹明甲酯（TMRM）用于观察线粒体膜电位（Δψ），碘化丙啶用于观察细胞活力丧失情况。在对培养的肝细胞进行化学性缺氧的最初30至40分钟内，形成了大量表面气泡，细胞体积增大，但线粒体膜电位下降相对较小。随后，线粒体膜的非特异性通透性增加，线粒体去极化。几分钟后，单个溶酶体解体。再过几分钟后，出现气泡破裂、钙黄绿素对质膜的总体通透性增加以及碘化丙啶对细胞核的标记，表明细胞失去活力。因此，在化学性缺氧过程中发生了以下细胞内事件序列：三磷酸腺苷（ATP）耗竭、细胞肿胀并形成气泡、线粒体通透性转变开始、溶酶体解体、气泡破裂导致质膜功能障碍以及细胞死亡。任何对缺氧损伤病理生理学的解释都必须考虑到这一独特的事件序列。
