Development of novel graft preservation system through functinal modulation of hepatic sinusoidal cel

1)建立了从大鼠肝脏分离枯否细胞、内皮细胞和Ito细胞的方法和技术。分离的细胞纯度大于90%。这为以下研究项目做出了重要贡献。2）我们揭示了库普弗细胞在移植肝冷藏和再灌注过程中的激活。激活的枯否细胞产生多种介质，包括TNF，其导致移植物损伤。抗肿瘤坏死因子治疗可有效预防长期冷保存后移植肝的损伤。我们进行了进一步的研究，以阐明肿瘤坏死因子依赖性移植物损伤的确切机制，表明激活的枯否细胞诱导内皮细胞和枯否细胞本身的凋亡，通过阿曲他克林和/或autocine机制。在激活的枯否细胞膜上表达的膜结合TNF可能参与了该事件。我们设计了反义DNA寡聚体，其被设计为靶向编码大鼠TNF-α的mRNA的各个位点。反义寡聚体特异性抑制库普弗细胞的TNF表达。因此，反义技术似乎有希望调节枯否细胞的功能。3）原位肝移植后发生微循环障碍。使用在体显微镜系统，我们能够证明，血管活性物质，内皮素-1，导致血管收缩在肝微血管。内皮素-1在缺血/再灌注和肝移植后升高。抗内皮素-1治疗可减轻原位肝移植术后移植物损伤。我们进一步阐明，窦张力是由两种血管活性物质，内皮素-1和一氧化氮之间的平衡调节。我们的研究还表明，内源性NO作为一种血管扩张剂，减弱内皮素诱导的血管收缩，从而改善微循环。这一结果提出了肝血流在正弦水平上调节的新概念。

1) We have established the methods and technology to separate Kupffer cells, endothelial cells and Ito cells from rat liver. The purity of the isolated cells are more than 90%. This contributed much to the follwing research projects.2) We revealed that Kupffer cells become activated during cold storage of liver grafts and reperfusion. Activated Kupffer cells produce variety of mediators including TNF, which causes damage to the grafts. Anti-TNF therapy was shown to be effective to prevent the liver damge after prolonged cold graft storage. We conducted further study to elucidate the precise mechanism of the TNF-dependent graft damage, showing that activated Kupffer cells induce apoptosis in endothelial cells and Kupffer cells themselves by juxtacrine and/or autocine mechanisms. Membrane-bound TNF expressed on the membrane of activated Kupffer cells is likely involved in this event. We enginnered antisense DNA oligomers which were designed to target various sites of mRNA encoding rat TNF-alpha. The antisense oligomers specifically suppressed TNF expression by Kupffer cells. Thus, the antisense technology appears to be promising to modulate Kupffer cell function.3) Microcirculatory disturbance occurs after orthotopic liver transplantation. Using an in vivo microscopy system, we were able to demonstrate that a vasoactive substance, endothelin-1, causes vasoconstriction in hepatic microvasculature. Endothelin-1 was shown to elevate after ischemia/reflow and liver transplantation. Anti-endothelin-1 therapy reduced the graft damage after orthotopic liver transplantation. We further elucidated that sinusoidal tone is regulated by the balance between two vasoactive substances, endothelin-1 and nitric oxide. Our study also suggests that endogenous NO acts as a vasodilator which attenuates endothelin-induced vasoconstriction, thereby improving microcirculation. The results indicate a novel concept that hepatic blood flow is regulated at the sinusoidal level.