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

背景。在肝缺血-再灌注损伤（IRI）中，血栓调节素（TM）从窦内皮细胞大量丢失被认为在肝内高凝性和肝损伤的发展中起核心作用。对肝移植物进行基因修饰以表达外源性TM可能有助于取代任何丢失的TM，从而减少肝脏IRI。材料和方法。三组12只大鼠分别静脉注射标记物LacZ腺病毒载体（AxCALacZ,1×10^9 cfu/ml）、人TM （hTM）腺病毒载体（AxCAhTM,1×10^9 cfu/ml）、生理盐水（1 ml）。基因转染48h后，用乙醚麻醉大鼠，肝热缺血30min。各组于再灌注后6、12、24、7 d各处死3只大鼠，取血清和肝组织标本。血清样本用于测定肝细胞酶（A…More LT）、透明质酸等的水平。用X-gal染色法检测标记物LacZ在肝脏的表达。采用免疫组化染色和RT-PCR检测hTM蛋白和mRNA的表达。用激光血流仪测量局部肝血流。结果在再灌注7 d后，1组和2组肝脏中分别检测到标记物LacZ （X-gal染色）和hTM（免疫组织化学染色和RT-PCR）的表达。再灌注后的肝内高凝性，如中心小静脉血栓所示，仅在再灌注后6至12小时内，hTM转染组2的肝内高凝性显著降低。再灌注后12 h，仅hTM治疗2组血清ALT水平、透明质酸、肝组织血流量、肝内中性粒细胞聚集较1、3组明显改善。这些结果提示，腺病毒介导的热媒基因转移有助于减轻热缺血性肝损伤大鼠模型的肝损伤。少

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