Analysis of active oxygen metabolism in organ microcirculation using confocal laser microscopy

建立了用激光共聚焦扫描成像系统进行活体观察的实验装置。我们的实验是用荧光探针进行的；二氯荧光素二醋酸酯（一种对细胞内过氧化氢形成敏感的荧光染料），罗丹明123（一种对线粒体膜电位敏感的荧光染料）和碘化丙啶（标记非活细胞的细胞）。利用上述装置；缺血再灌注后大鼠肠系膜微循环出现白细胞粘附、氧化应激和线粒体功能障碍。在灌注肝低流量缺氧时，氧化应激和线粒体功能障碍的小叶内异质性与细胞死亡的地形分布之间的关系被可视化。低流量缺氧时的氧化应激和由此引起的细胞死亡在空间上仅限于门静脉周围和中央周围之间的中间区域。冷缺血后再灌注诱导白细胞粘附于窦壁上，损伤实质细胞。超氧化物歧化酶可减轻缺血再灌注引起的损伤。观察肿瘤细胞与Kupffer细胞共培养后的代谢变化。在共培养的肝癌细胞和结肠癌细胞中，Kupffer细胞诱导氧化应激和线粒体功能障碍，随后导致细胞死亡。超氧化物歧化酶对线粒体功能障碍和细胞死亡无抑制作用。一氧化氮生成抑制剂L-NMMA可消除Kupffer细胞诱导的线粒体能量减少和肿瘤细胞损伤。Kupffer细胞来源的一氧化氮可能通过抑制线粒体功能障碍而呈现肿瘤细胞毒性。体外灌注肝后肿瘤细胞的代谢变化也被测定。粘附于肝窦的肿瘤细胞氧化应激和线粒体功能障碍与体外实验一样明显。少

We have established the experimental set up for intravital observation using confocal laser scanning imaging system. Our experiments were performed using fluorescence probes ; dichlorofluorescin diacetate (a fluorochrome sensitive to intracellular hydroperoxide formation), rhodamine 123 (a fluorochrome sensitive to the mitochondrial membrane potential, ) and propidium iodide (which labels the unclei of non viable cells).By using above mentioned set up ;1. It has been observed that leukocyte adhesion along postcapillary venules, the oxidative stress and the mitochondrial dysfunction occur in rat mesenteric microcirculation after ischemiareperfusion.2. Intralobular heterogeneity of the oxidative stress and the mitochondrial dysfunction were visualized in relation to their topographic distributions with the cell death during low-flow hypoxia in perfused liver. The oxidative stress and the resultant cell death during low-fow hypoxia are spatially restricted in the intermediate zone between … More the periportal and pericentral regions.3. Cold ischemia followed by reperfusion induced leukocyte adhesion to the sinusoidal wall and the damage of parenchymal cells. Superoxide dismutase attenuated these ischemiareperfusion-induced injuries.4. The metabolic changes in tumor cells after coculturing with Kupffer cells were visualized and analyzed. In cocultured hepatoma cells and colon cancer cells, Kupffer cells induced the oxidative stress and the mitochondrial dysfunction followed by cell death. Superoxide dismutase did not inhibit the mitochondrial dysfunction and cell death. L-NMMA,an inhibitor of nitric oxide production eliminated decrease in mitochondrial energization and the tumor cell injury induced by Kupffer cells. Kupffer cell-derived nitric oxide may present tumor cell cytotoxicity via inhibiting mitochondrial dysfunction.The metabolic changes in tumor cells were also determined ex vivo perfused liver. The oxidative stress and the mitochondrial dysfunction in tumor cells, which adhered to the liver sinusoid, were also obvious as well as those in vitro experiments. Less