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.

本研究的目的是阐明缺血再灌注（I/R）损伤或小尺寸移植物（SSG）损伤的机制，并利用基因转移技术创建抗各种损伤的超级肝移植物。 (1) 基因转移技术的发展：使用专为大鼠肝移植物设计的新仪器，将电穿孔介导的基因转移到冷保存的移植物中，而不是具有白血病严重风险的逆转录病毒载体(Science 2002)。使用电穿孔和流体动力（=门静脉正压）技术的组合方法，发现肝移植后移植物中荧光素酶基因的表达增加。但也观察到明显的移植物损伤。因此，现在应用了一种使用纳米载体的新基因转移方法。（2）使用小型移植物的肝移植（LT）模型：在使用小型移植物（SSG）的新型大鼠LT模型中：整个肝脏体积的30%），建议通过部分门体分流（皮下脾固定术）或施用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存活时间。因此，SSG门脉灌注过度被证明是SSG损伤的重要原因。并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相关的分子物质，被发现在缺血再灌注损伤中发挥着重要作用。 /R伤害。其抑制剂（Fasudil）可抑制 I/R 损伤中细胞因子的产生（TNFa 和 IL1b）和自由基的产生。通过给予法舒地尔，术后 AST 和 ALT 升高以及标本中的坏死区域减少，此外，在 24 小时冷保存 LT 模型中，LT 后大鼠的存活率提高。现在使用非病毒基因转移技术将 Rho 激酶基因的反义或显性失活应用于肝移植。