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

通过肝窦细胞功能调节开发新型移植物保存系统

基本信息

批准号：
04454244
负责人：
KAWANO Sunao
金额：
$ 4.35万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

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.

1）建立了从大鼠肝脏中分离Kupffer细胞、内皮细胞和Ito细胞的方法和技术。分离的细胞纯度达到90%以上。这对后续的研究项目做出了很大贡献。2）我们发现库普弗细胞在肝移植物的冷藏和再灌注过程中被激活。激活的库普弗细胞产生多种介质，包括肿瘤坏死因子，这会对移植物造成损害。抗肿瘤坏死因子治疗被证明可以有效预防移植物长期冷藏后的肝脏损伤。我们进行了进一步的研究，以阐明 TNF 依赖性移植物损伤的精确机制，表明活化的 Kupffer 细胞通过近分泌和/或自分泌机制诱导内皮细胞和 Kupffer 细胞本身凋亡。在活化的库普弗细胞膜上表达的膜结合 TNF 可能参与了这一事件。我们设计了反义 DNA 寡聚物，旨在靶向编码大鼠 TNF-α 的 mRNA 的各个位点。反义寡聚物特异性抑制库普弗细胞的 TNF 表达。因此，反义技术似乎有望调节库普弗细胞功能。3)原位肝移植后发生微循环障碍。使用体内显微镜系统，我们能够证明血管活性物质内皮素-1 会引起肝微血管系统的血管收缩。缺血/复流和肝移植后，内皮素-1 升高。抗内皮素-1治疗减少了原位肝移植后的移植物损伤。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 作为血管舒张剂，可以减弱内皮素诱导的血管收缩，从而改善微循环。结果表明了一个新概念，即肝血流在正弦水平上受到调节。