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

1995年，我们建立了能够检测一氧化氮(NO)和肿瘤坏死因子-α(TNF-α)的产生以及DNA片段化和核改变的凋亡肝癌细胞的系统。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的mRNAs的表达。研究表明，Kupffer细胞与肝癌细胞共培养可刺激NO和TNF-α的产生，Kupffer细胞释放的这些介质协同诱导肝癌细胞的凋亡。使用针对iNOS和TNF-α的反义和正义寡核苷酸以及针对黏附分子的抗体的抑制研究表明，通过CD18和ICAM-1的相互作用导致与肝癌细胞共同培养的Kupffer细胞的激活和介质产生。我们进一步建立了检测活化的核因子-kappaB的方法(荧光原位DNA-蛋白结合分析)。本实验证实CD18与ICAM-1结合可激活核因子-kappaB。

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.