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Development of a super liver graft resistant to various kinds of injuries by a gene-transfer technique

通过基因转移技术开发出抗各种损伤的超级肝移植物

基本信息

批准号：
13357011
负责人：
SHIMADA Mitsuo
金额：
$ 32.86万
依托单位：
Kyushu University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

项目摘要

This study was conducted to clarify the mechanism of ischemia-reperfusion (I/R) injury or small-for-size graft (SSG) injury, and to create a super liver graft resistant to various kinds of injuries using a gene-transfer technique.(1) Development of gene-transfer technique :Instead of retrovirus vector with critical risk of leukemia (Science 2002), electroporation-mediated gene transfer to cold preserved graft was carried out, using a new instrument designed for a rat liver graft. Using a combined method of electroporation and hydrodynamic (=positive pressure to the portal vein) technique, luciferase-gene expression in the graft after liver transplantation was found to be increased. But a significant graft damage was also observed. A new gene-transfer method using nano-carrier is, therefore, now applied.(2) Liver transplant (LT) model using a small-for-size graft :In a new rat LT model using small-for-size graft (SSG : 30% of whole liver volume), survival of transplanted rats was suggested to prolong either by either creation of partial porto-systemic shunt (splenopexy beneath the skin) or administration of somatostatin analogue having effect of reduced portal pressure. Therefore, excessive portal perfusion of SSG was proved to be a critical cause of SSG injury. A new LT model was, furthermore, completed in mice.(3) Role of Rho-kinage on ischemia-reperfusion injury :Rho-kinase, a molecular substance related to vascular contraction or neutrophil migration, was found to played an important role on I/R injury. Its inhibitor (Fasudil) inhibited cytokine production (TNFa and IL1b) and free radical production in I/R injury. By administration of Fasudil, postoperative elevation of AST and ALT, and necrotic areas in the specimen, were reduced, furthermore, rat's survival rate after LT improved in a 24-hour cold-preserved LT model. Either anti-sense or dominant negative of Rho-kinase gene is now applied to liver graft using a non-viral gene-transfer technique.

本研究旨在阐明缺血-再灌注（I/R）损伤或小体积移植物（SSG）损伤的机制，并利用基因转移技术建立抗各种损伤的超级肝移植物。(1)基因转移技术的发展：使用为大鼠肝移植物设计的新仪器，进行电穿孔介导的基因转移至冷保存移植物，而不是具有白血病临界风险的逆转录病毒载体（Science 2002）。使用电穿孔和流体动力学（=门静脉正压）技术相结合的方法，发现肝移植后移植物中的胰蛋白酶基因表达增加。但也观察到明显的移植物损伤。因此，一种新的基因转移方法，使用纳米载体，现在应用。(2)使用小体积移植物的肝移植（LT）模型：在使用小体积移植物（SSG：整个肝脏体积的30%）的新大鼠LT模型中，建议通过建立部分门体分流（皮下脾固定术）或给予具有降低门静脉压力作用的生长抑素类似物来延长移植大鼠的生存期。因此，门静脉SSG过度灌注是导致SSG损伤的重要原因。此外，在小鼠中完成了新的LT模型。(3)Rho激酶在缺血再灌注损伤中的作用：Rho激酶是一种与血管收缩或中性粒细胞迁移有关的分子物质，在I/R损伤中起重要作用。其抑制剂法舒地尔（Fasudil）可抑制I/R损伤中细胞因子（TNF α和IL 1b）的产生和自由基的产生。法舒地尔可降低肝移植术后AST、ALT的升高，减少肝移植术后坏死面积，提高大鼠24小时冷保存肝移植后的存活率。Rho激酶基因的反义或显性失活是目前应用于肝移植的非病毒基因转移技术。