Fukumura D, Yonei Y, Kurose I, Saito H, Ohishi T, Higuchi H, Miura S, Kato S, Kimura H, Ebinuma H, Ishi H
Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Hepatology. 1996 Jul;24(1):141-9. doi: 10.1053/jhep.1996.v24.pm0008707254.
The metabolic change in tumor cells (AH70, a rat hepatoma cell line) cocultured with isolated rat Kupffer cells were visualized and analyzed by a laser scanning confocal imaging system. When AH70 cells were cocultured with Kupffer cells, fluorescence intensity of rhodamine 123 (Rh123) decreased, indicating the reduction of mitochondrial function. The reduction in Rh123 was eliminated by NG-monomethyl-L-arginine (L-NMMA), an analogue of L-arginine, suggesting the involvement of nitric oxide (NO). Two hour after the cells were cocultured, membrane compromised AH70 cells which were observed as propidium from 2.8% to 25%. This increase was also attenuated by L-NMMA, suggesting that Kupffer cell-mediated injury of tumor cells largely depends on NO. The concentrations of NO-2 + NO-3 in the culture medium markedly increased after coculture of AH70 cells with Kupffer cells. Moreover, NO synthase (NOS) activity in Kupffer cells significantly increased after coculture. These in vitro results suggest that NO mediates Kupffer cell-induced tumor cell damage characterized by reduced mitochondrial function and diminished barrier function. In the ex vivo study of the perfused liver to which AH70 cells were injected via the catheter inserted into the portal vein, some AH70 cells were arrested in the upper stream of sinusoid and the fluorescence intensity of Rh123 in adherent AH70 cells decreased in a time-dependent manner within 2 hours. The number of PI-positive AH70 cells also increased 2 hours after the injection of AH70 cells. These changes were inhibited by either administration of N omega-L-nitroarginine-methylester (L-NAME) to perfusate or pretreatment of the rat liver with GdCl3, which is known to deplete Kupffer cell function. Thus, the present study suggests that NO from Kupffer cells induces mitochondrial dysfunction in tumor cells followed by membrane barrier dysfunction in the liver sinusoid.
利用激光扫描共聚焦成像系统对与分离的大鼠库普弗细胞共培养的肿瘤细胞（AH70，一种大鼠肝癌细胞系）的代谢变化进行可视化和分析。当AH70细胞与库普弗细胞共培养时，罗丹明123（Rh123）的荧光强度降低，表明线粒体功能下降。L-精氨酸类似物NG-单甲基-L-精氨酸（L-NMMA）消除了Rh123的降低，提示一氧化氮（NO）参与其中。细胞共培养2小时后，膜受损的AH70细胞从2.8%增加到25%，这一增加也被L-NMMA减弱，表明库普弗细胞介导的肿瘤细胞损伤很大程度上依赖于NO。AH70细胞与库普弗细胞共培养后，培养基中NO₂ + NO₃的浓度显著增加。此外，共培养后库普弗细胞中的一氧化氮合酶（NOS）活性显著增加。这些体外研究结果表明，NO介导了库普弗细胞诱导的肿瘤细胞损伤，其特征为线粒体功能降低和屏障功能减弱。在通过插入门静脉的导管向灌注肝脏注射AH70细胞的离体研究中，一些AH70细胞停滞在肝血窦上游，贴壁的AH70细胞中Rh123的荧光强度在2小时内呈时间依赖性降低。注射AH70细胞2小时后，PI阳性的AH70细胞数量也增加。向灌注液中给予Nω-L-硝基精氨酸甲酯（L-NAME）或用已知可耗尽库普弗细胞功能的GdCl₃预处理大鼠肝脏均可抑制这些变化。因此，本研究表明，库普弗细胞产生的NO诱导肿瘤细胞线粒体功能障碍，随后导致肝血窦膜屏障功能障碍。
