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

1)建立了从大鼠肝脏分离Kupffer细胞、内皮细胞和Ito细胞的方法和技术。分离的细胞纯度在90%以上。这为后续的研究项目做出了很大贡献。2)我们发现Kupffer细胞在移植肝的冷保存和再灌流过程中被激活。激活的库普弗细胞产生包括肿瘤坏死因子在内的多种介质，对移植物造成损害。抗肿瘤坏死因子治疗能有效预防移植肝冷保存后的肝损伤。我们进一步研究了肿瘤坏死因子依赖的移植物损伤的确切机制，发现激活的Kupffer细胞通过旁分泌和/或自体电影机制诱导内皮细胞和Kupffer细胞本身的凋亡。活化的库普弗细胞膜上表达的膜结合的肿瘤坏死因子可能参与了这一事件。我们设计了反义DNA寡聚体，目的是针对编码大鼠肿瘤坏死因子-α的不同位置的mRNA。反义寡聚物可特异性抑制枯否细胞表达肿瘤坏死因子。因此，反义技术似乎有望调节Kupffer细胞的功能。3)原位肝移植后出现微循环障碍。使用活体显微镜系统，我们能够证明一种血管活性物质，内皮素-1，引起肝脏微血管的血管收缩。缺血/复流和肝移植后ET-1升高。抗内皮素-1治疗减轻了原位肝移植后移植物的损伤。我们进一步阐明，正弦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的。我们的研究还表明，内源性NO具有血管扩张剂的作用，可以减弱内皮素诱导的血管收缩，从而改善微循环。结果表明，肝脏血流是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.