肝动脉缺血对肝移植术后胆管树纤维化影响的机理及防治措施

Mechanism of Effect of Hepatic Artery Ischemia on Biliary Fibrosis after Liver Transplantation and Prevention Method

目的探讨肝动脉缺血对肝移植术后胆管树纤维化影响的机理及防治措施。方法 18只雄性成年实验犬被制成简易自体原位肝移植模型并随机分为肝动脉缺血组(HAI组)、肝动脉桥式置管转流组(TBB组)及对照组,每组6只。3组动物于门静脉开放后6 h、3 d和14 d时切取部分肝组织,观察肝内胆管形态学变化,用免疫组化法检测肝内胆管上皮细胞中转化生长因子(TGF)-β1蛋白的表达;并于14 d时活杀动物,采用Western blot法测定肝内胆管组织中Smad3及磷酸化Smad3蛋白表达水平,RT-PCR法测定肝内胆管组织中α-平滑肌肌动蛋白(SMA)mRNA转录水平。结果与对照组比较,HAI组中可观察到明显的肝内胆管上皮细胞增生,胶原蛋白沉积增多,胆管管腔狭窄;而TBB组肝内胆管的形态学病理改变轻于HAI组。TBB组TGF-β1阳性细胞指数在门静脉开放后3 d时达到峰值,随后下降;而HAI组则持续升高,并明显高于TBB组(P<0.05)。在门静脉开放后14 d时,TBB组磷酸化Smad3蛋白表达水平和α-SMA mRNA的转录水平分别为1.04±0.13和1.12±0.55,显著高于对照组(0.59±0.09和0.46±0.18),但低于HAI组(1.82±0.18和1.86±0.73),3组之间差异有统计学意义(P<0.05);而3组间Smad3蛋白的表达水平差异无统计学意义(P>0.05)。结论肝动脉缺血通过对TGF-β1/Smads信号转导通路的活化,引起肝内胆管壁中胶原纤维的沉积,肌成纤维细胞转化的增多,导致胆管树纤维化的发生;肝动脉桥式置管转流装置能够通过抑制TGF-β1/Smads信号转导通路活化来减轻肝动脉缺血引起的胆管树纤维化。

Objective To validate the mechanism of effect of hepatic artery ischemia on biliary fibrosis after liver transplantation and the prevention method.Methods Eighteen male dogs were established into the concise auto orthotopic liver transplantation models and assigned into three groups randomly： hepatic artery ischemia（HAI） group,TBB group（transferred the blood by a bridge duct） and control group,each group contained 6 dogs.After opening portal vein,the samples were cut from liver in each group at the time of 6 h,3 d and 14 d.The pathological modifications of intrahepatic bile ducts were observed and expression of transforming growth factor-β1（TGF-β1） were detected in the three times.Expressions of Smad3 and phosphate-Smad3 as well as mRNA of α-smooth muscle actin（α-SMA） in intrahepatic bile ducts were detected 14 d after opening portal vein.Results Compared with control group,the collagen deposition and lumens stenosis in biliary vessel wall were more obviously in HAI group.In TBB group,the pathological modifications were slighter compared with HAI group.The positive cell index of TGF-β1 reached peak on day 3 after opening portal vein,then decreased in TBB group,and which in HAI group kept increase and was significantly higher than that in TBB group（P〈0.05）.The expression level of phosphate-Smad3 and transcriptional level of α-SMA mRNA were 1.04±0.13 and 1.12±0.55 in TBB group on day 14 after opening portal vein,which were significantly higher than those in control group（0.59±0.09 and 0.46±0.18） and lower than those in HAI group（1.82±0.18 and 1.86±0.73）,the diversities among three groups were significant（P〈0.05）.There was not significant difference of expression of Smads among three groups（P〈0.05）.ConclusionsHepatic artery ischemia could increase the deposition of collagen fibers and the transdifferentiation of myofibroblast in bile duct and result in the biliary fibrosis by activating the TGF-β1/Smads signaling pathway.The bridging bypass device could lessen the biliary fibrosis caused by hepatic artery ischemia by inhibiting the activation of TGF-β1/Smads signal transduction passageway.