In vivo gene transfer of human thrombomodulin improves intrahepatic hypercoagulability and ischemia-reperfusion injury after warm ischemia.

人血栓调节蛋白的体内基因转移可改善肝内高凝状态和热缺血后的缺血再灌注损伤。

• 批准号: 15591419
• 负责人: SHIRAISHI Masayuki
• 金额: $ 2.18万
• 依托单位: University of the Ryukyus
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591419/
• 关键词: thrombomodulin; ischemia-reperfusion injury; adenovirus vector; genetherapy; warm ischemia; non-heart beating donor; 遺伝子冶療; 肝臓移植; 心臓死

Background.In hepatic ischemia-reperfusion injury (IRI), a massive loss of thrombomodulin (TM) from the sinusoidal endothelial cells is thought to play a central role in the development of intrahepatic hypercoagulability and liver damage. The genetic modification of the liver graft to express exogenous TM might thus help to replace any lost TM, thereby reducing hepatic IRI.Materials and methods.Three groups of 12 rats each received an intra-venous injection of marker LacZ adenovirus vector in group 1 (AxCALacZ,1×10^9 cfu/ml), human TM (hTM) adenovirus vector in group 2 (AxCAhTM,1×10^9 cfu/ml), or normal saline in group 3 (1 ml). Forty-eight hrs after gene transfer, the rats were anesthetized by ether and then were subjected to hepatic warm ischemia for 30 min. In all the groups, 3 rats each were sacrificed at 6, 12, 24 hrs, and 7 days after reperfusion, in order to obtain both serum and hepatic tissue samples. The serum samples were used to determine the levels of hepatocyte enzymes (A … More LT), hyaluronic acid, and others. The hepatic expression of marker LacZ was evaluated by X-gal staining. The protein and mRNA expression of hTM were evaluated by immunohistochemical staining and RT-PCR. The local hepatic blood flow was measured by a laser blood flowmeter. Intrahepatic neutrophil aggregation was also evaluated by Naphthol AS-D chloroacetate staining.Results.The expression of marker LacZ (X-gal staining ) and hTM (immunohistochemical staining and RT-PCR) was detected only in the livers from groups 1 and 2, respectively, up until 7 days after reperfusion. Intrahepatic hypercoagulability after reperfusion, as indicated by thrombus in the central venules, only decreased remarkably in the hTM transfected group 2 at 6 to 12 hrs after reperfusion. At 12 hrs after reperfusion, the serum ALT levels, hyaluronic acid, hepatic tissue blood flow, and intrahepatic neutrophil aggregation significantly improved only in the hTM treated group 2 in comparison to those of groups 1 and 3.Conclusions.These findings thus suggested that the adenovirus mediated gene transfer of hTM helped to attenuate liver damage in a rat model of warm ischemic liver injury. Less

背景：在肝缺血再灌注损伤（IRI）中，血窦内皮细胞血栓调节蛋白（TM）的大量丢失被认为在肝内高凝状态和肝损伤的发展中起重要作用。因此，对肝移植物进行基因修饰以表达外源性TM可能有助于替换任何丢失的TM，从而降低肝IRI。（AxCALacZ，1×10^9 cfu/ml），组2中的人TM（hTM）腺病毒载体（AxCAhTM，1×10^9 cfu/ml），或组3中的生理盐水（1 ml）。基因转移后48小时，大鼠麻醉乙醚，然后进行肝脏热缺血30分钟。在所有组中，3只大鼠分别在6，12，24小时，7天后再灌注，以获得血清和肝组织样品。血清标本用于测定肝细胞酶（A ...更多信息 LT）、透明质酸等。通过X-gal染色评估标志物LacZ的肝表达。采用免疫组化和RT-PCR方法检测hTM蛋白和mRNA的表达。用激光血流量计测量局部肝血流量。肝内中性粒细胞聚集也进行了评估，Naphthalocyanas-D chloroacetate staining.Results的标记LacZ（X-gal染色）和hTM（免疫组化染色和RT-PCR）的表达仅在肝脏检测组1和2，分别，直到7天后再灌注。再灌注后的肝内高凝状态，如中央小静脉中的血栓所示，仅在再灌注后6至12小时在hTM转染组2中显著降低。再灌注12 h后，hTM治疗组2血清ALT、透明质酸、肝组织血流量、肝内中性粒细胞聚集率均较治疗组1和3明显改善。结论腺病毒介导的hTM基因转染可减轻大鼠热缺血性肝损伤。少