【作者】 梁靓；

【导师】 梁廷波；

【作者基本信息】 浙江大学 ， 外科学， 2009， 博士

【摘要】 实验目的:对小体积肝移植的基础研究发现移植术后存在门静脉高灌注状态并导致早期细胞内稳态改变从而引起移植物损伤加重,但术后晚期或长期移植物状态特别是肝窦内微循环特点仍未明确。本实验在建立大鼠小体积肝移植模型基础上研究长期移植物肝窦微循环状态及内在的细胞调节作用。材料与方法:根据供肝体积建立大鼠小体积肝移植组(实验组)及全肝移植组(对照组),观察7天生存情况;检测术中及术后1小时内的门静脉压力;利用活体荧光显微镜观察移植物微循环状态;通过实时定量PCR、免疫蛋白印记等方法检测肝内与肝星状细胞活化相关蛋白、内皮素-1及其受体表达情况并并在光镜及透射电镜下观察移植物病理变化。此外,建立20对大鼠小体积肝移植模型并分成2组:于受体门静脉吻合开放即刻于门静脉分支处分别注射BQ-123或BQ-788,于术后检测微循环并取血清标本行生化检测。结果:(1)实验组7天生存率为50%,明显低于对照组(100%);(2)实验组血清ALT、AST水平在术后1天达到高峰且高于对照组,尽管1天后有所回落但仍持续高于对照组;并在术后3天起血清TB水平高于对照组;(3)实验组受体术后1h内门静脉压力要显著高于对照组,但此后两组门静脉压力均恢复到基础水平,无明显差异;(4)实验组术后肝窦微循环状态明显恶化,包括肝窦红细胞流速持续升高;肝窦再灌注情况明显降低;肝窦出现一定程度的扩张;肝细胞凋亡增加;肝细胞的核间距增大;在电镜及光镜检查中出现不可逆的病理改变;(5)实验组术后HSC活化增强,表现为:自发荧光减少、活化相关蛋白表达上调、形态改变等;(6)实验组术后内皮素-1及其受体(尤其是A型受体)表达升高,且受体主要表达于活化的HSC内而其他肝内细胞如肝细胞或内皮细胞表达较少;(7)小体积肝移植术后加用BQ-123能改善肝窦微循环,而术后加用BQ-788对肝窦微循环的影响不明显。结论:在小体积肝移植术后虽然门静脉压力在1小时以后恢复正常,但肝窦微循环的恶化是持续存在,并可能是导致移植物晚期损伤的重要机制。肝星状细胞在小体积肝移植术后发生明显的活化,胞浆内产生大量与细胞收缩相关的平滑肌肌动蛋白等纤维束提示该细胞对于术后微循环的调节发挥了一定的作用。术后高内皮素水平及肝内A受体表达的升高提示HSC通过细胞表面的A型受体收缩临近肝窦,从而发挥术后对肝窦直径及血流的自身调节作用。BQ-123能选择性阻断内皮素与其A型的作用发挥改善移植物微循环状态的作用。BQ-788的作用不明显也提示在B型受体通路在移植物损伤中并非关键因素。更多还原

【Abstract】 Aim:Small-for-size graft injury is characterized by portal venous hyperperfusion and loss of intracellular homeostasis early after transplant.The long-term alteration of sinusoidal microcirculation and the auto-regulatory factor on graft function remain unknown.An experimental rat model of syngeneic liver transplantation with small-for-size and whole grafts was designed to examine:(1) whether microcirculatory disturbance recovers after portal pressure returns to baseline;(2) whether graft injury after liver transplantation leads to HSC activation with specific gene expression;(3) the response of sinusoidal auto-regulation to graft injury,especially after small-for-size liver transplantation;and(4) the role of different antagonist of ET receptor subtype on sinusoidal microcirculatory on small-for-size graft.Methods and method:An experimental rat model with small-for-size and whole grafts was designed. survival and portal pressure were detected.Sinusoidal microcirculation was examined by intravital fluorescence microscopy after each timepoint after transplantation.HSC activation-related protein expression was determined by real-time quantitative reverse-transcriptase polymerase chain reaction,Western blots and immunohistochemistry. Endothelin-1 and its receptors,and ultrastructural changes were also examined.Results:Although portal hypertension only persisted for 1 hour,a sustained microcirculatory hyper-hemodynamic state was accompanied by reduction of sinusoidal perfusion as well as elevation of sinusoidal diameter and number of apoptotic hepatocytes during the first 7 days.These resulted in lower survival rate and graft dysfunction.More hepatic stellate cells were subject to activation and transformation into myofibroblast-like cells from 1 day after transplantation.Serum endothelin-1 levels were significantly increased after transplantation,accompanied by over-expression of endothelin-1 A receptors on hepatic stellate cells.Sinusoidal derangement was attenuated by BQ-123,a antagonist of ETAR while no significant changes were showed in BQ-788 group.Conclusions:Small-for-size liver transplantation displayed progressive graft injury including long-term microcirculation disturbance,hepatocyte apoptosis and hepatic stellate cell activation,although portal hypertension transiently persisted for only 1 hour after reperfusion.Endothelin-1 might modulate sinusoidal microcirculation mostly by endothelin-1 A receptor-mediated hepatic stellate cell constriction in both nonperfused and perfused sinusoid with different effects.BQ-123,as a antagonist of ETAR could attenuate sinusoidal microcirculation by increasing the reperfusion rates,decreasing attenuate sinusoidal microcirculation by increasing the reperfusion rates,decreasing velocity and sinusoidal diameter rather than reducing hepatocytes apoptosis.However, BQ-788,which is a antagonist of ETBR didn’t exert significant protective role on graft injuries.更多还原