Mechanism through which Kupffer cell-derived active oxidants mediate hepatoma cell injury

在1995年，我们建立了一个系统，可以确定一氧化氮（NO）和TNF-α的生产和凋亡肝癌细胞的DNA片段和核改变。1996年，我们利用激光扫描共聚焦显微镜，成功地观察和分析了诱导型一氧化氮合酶（iNOS）和肿瘤坏死因子-α（TNF-α）mRNA的表达。使用这些技术的研究表明，库普弗细胞和肝癌细胞的共培养刺激NO和TNF-α的产生，这些介质从库普弗细胞释放协同诱导肝癌细胞的凋亡。使用反义和正义寡脱氧核苷酸对iNOS和TNF-α mRNA的抑制剂研究，对粘附分子的抗体表明，通过CD 18和ICAM-1的相互作用导致与肝癌细胞共培养的库普弗细胞的激活和介体产生。我们进一步建立了激活的NF-κ B可以可视化的方法（荧光原位DNA-蛋白结合测定）。通过该测定，已经证明CD 18和ICAM-1之间的结合导致NF-κ B的活化。

In 1995, we established the system which can determine productions of nitric oxide (NO) and TNF-alpha and apoptotic hepatoma cells with fragmented DNA and nuclear alterations. In 1996, by using laser scanning confocal microscope, we succeeded to visualize and analyze expression of mRNAs of inducible NO synthase (iNOS) and TNF-alpha. Investigations using these techniques revealed that coculture of Kupffer cells and hepatoma cells stimulate productions of NO and TNF-alpha, and these mediators released from Kupffer cells synergistically induce apoptosis of hepatoma cells. Inhibitor studies using antisense and sense oligodeoxynucleotides against mRNAs of iNOS and TNF-alpha, antibodies against adhesion molecules suggested that interactions via CD18 and ICAM-1 leads to activation and mediator production of Kupffer cells cocultured with hepatoma cells. We further established the method by which activated NF-kappaB can be visualized (Fluorescence in situ DNA-protein binding assay). By this assay, binding between CD18 and ICAM-1 have been demonstrated to lead to activation of NF-kappaB.