【作者】 程龙；

【导师】 王曙光；

【作者基本信息】 第三军医大学 ， 外科学， 2010， 博士

【摘要】 一、研究背景胆道并发症仍然是严重影响肝移植受者生活质量及长期生存率的并发症之一。随着外科技术的进步,吻合口狭窄等并发症呈逐渐下降的趋势,而非吻合口并发症,如缺血型胆道病变,则成为肝移植术后胆道并发症的主要临床病理表现。肝脏移植术后缺血型胆道病变的发生机制尚不明确,目前,学者普遍认为与胆道缺血、免疫等因素有关。近年来,越来越多的研究表明胆汁中胆盐/磷脂比升高在肝移植后胆管损伤中起着重要作用。然而,肝移植后胆盐/磷脂比升高引起胆管及其上皮细胞损伤的具体机制尚无研究报道。研究表明,胆盐除了有“去污作用”外,还可在细胞内发挥信号分子的作用,参与细胞的增生和凋亡;而且由于其“去污作用”需要在高浓度下才能发挥,所以胆盐以信号分子的角色参与胆管细胞的病理生理改变可能更为普遍。在生理情况下,胆管细胞可经顶膜钠依赖性胆汁酸转运体(apical sodium-dependent bile acid transporter, ASBT)进行胆汁酸的重吸收,由细胞内胆汁酸结合蛋白(ileal bile acid binding protein, IBABP)转运至细胞基底侧,并经有机溶质转运体α/β(organic solute transporter alpha/beta, Ostα/β)等转出至门脉系统,从而启动了胆汁酸的“胆肝循环”。平衡的“胆肝循环”不会引起胆盐在细胞内潴留,这也是胆管细胞对胆盐毒性的一种自我保护机制。研究表明在多种炎症性及代谢性疾病中,胆汁酸转运蛋白表达发生改变,引起胆汁酸转运的紊乱,从而参与了疾病的发生发展。据以上观点和现象,我们推测,在移植肝冷保存再灌注损伤时,由于炎症因子的作用及核受体(如类法尼醇受体)表达/功能的改变,胆管细胞的胆汁酸转运平衡可能受到破坏,导致胆管细胞对胆盐毒性的自我保护功能紊乱,胆盐在胆管细胞内聚集,从而引起胆管细胞的损伤。二、研究目的基于以上假设,本研究以同系大鼠肝移植模型为研究对象,探讨移植肝胆管细胞胆盐转运蛋白在胆管损伤中的作用,并利用体外培养的胆管研究类法尼醇受体(Farnesoid X receptor, FXR)对胆管细胞胆汁酸转运蛋白表达的影响,探讨肝移植后胆管细胞胆盐转运蛋白参与胆管细胞损伤的机制。三、研究方法及结果1.利用免疫组织化学检测肝组织中ASBT、IBABP、Ostα及Ostβ的表达分布,结果显示,ASBT表达于大胆管细胞顶侧胞膜,IBABP表达于大胆管细胞浆,Ostα/Ostβ异二聚体表达于大、小胆管细胞基底侧胞膜及肝细胞血窦面胞膜。同时,我们用Real time PCR及Western blot方法检测分离的大小胆管组织中ASBT、IBABP及Ostα-Ostβ的表达,结果也显示,大胆管组织中ASBT和IBABP的表达显著高于小胆管,而Ostα-Ostβ在大胆管和小胆管组织中的表达量无显著差异。2.我们从mRNA水平和蛋白水平检测了移植肝胆管细胞胆盐转运蛋白的表达变化,并通过组织形态学观察和细胞凋亡检测评估移植肝胆管损伤的严重程度,结果显示,大鼠肝移植术后早期(术后1-3天)胆管细胞胆盐转运蛋白的表达均降低,降低幅度与供肝冷保存时间相关。随后,位于细胞顶侧的ASBT与位于胞浆内的IBABP及基底侧Ostα-Ostβ呈现不同的恢复速度及变化趋势,即ASBT很快恢复到正常水平,且在术后第7天其表达水平显著高于正常,而IBABP和Ostα-Osβ表达长时间降低,直到术后第7天(短冷却血组)甚至到第14天(长冷缺血组)才恢复到正常水平。ASBT与IBABP、Ostα和Osβ表达水平的比值与大胆管损伤严重程度显著相关。3.在检测移植肝胆汁中胆汁酸和磷脂浓度变化情况的基础上,我们通过免疫组织化学、荧光实时定量PCR、Western blotting及电泳移动漂移实验等技术,探讨不同冷保存时间下移植肝FXR表达及功能变化,结果显示,供肝冷保存再灌注损伤并不能引起胆汁中总胆汁酸的浓度的改变,只是通过降低磷脂的浓度,使胆汁酸/磷脂比增加,从而升高了胆汁中胆汁酸的相对浓度。大鼠肝移植后胆管组织中FXR的表达及功能活性显著降低,供肝冷保存时间越长,降低越明显。4.通过分离培养SD大鼠肝内胆管,探讨FXR对与胆汁共培养的胆管细胞之胆盐转运蛋白表达的影响,结果显示,在正常胆管上皮细胞中,毒性胆汁可促进ASBT、IBABP、Ostα及Ostβ的表达均增加;而在转染了FXR shRNA的胆管细胞中,毒性胆汁可同样升高ASBT的表达,而IBABP、Ostα和Ostβ的表达并未增加,反而下降。四、结论1.胆管细胞的胆汁酸转运主要发生在大胆管细胞。2.肝移植后胆管细胞胆汁酸转运蛋白表达出现不平行的变化,这种不平行改变程度与大胆管损伤严重程度显著相关。3.肝移植后毒性胆汁促进ASBT表达快速恢复甚至高于正常水平,而由于FXR表达及功能受到抑制,阻断了胆汁酸对IBABP、Ostα和Ostβ的促进作用,使IBABP、Ostα和Ostβ表达恢复缓慢,从而引起了胆管细胞胆盐转运蛋白的不平行改变。更多还原

【Abstract】 BackgroundBiliary complications remain the major source of morbidity for liver transplant recipients. Owing to the improvement of surgical techniques, the incidence of anastomotic strictures has continued to decrease, with the result that the non-anastomotic form is now the main type of biliary complication. Non-anastomotic strictures in the presence of a patent hepatic artery have been described as ischemic-type biliary lesions (ITBL), recently called ischemic cholangiopathy.The molecular mechanisms involved in the pathogenesis of ITBL remain unclear. Recent year, increasing number of studies have indicated that an altered bile composition is associated with bile duct injury and with the subsequent development of non-anastomotic biliary strictures following human liver transplantation. However, the molecular mechanism by which the altered bile induces injury to the bile duct epithelial cells remains to be elucidated. It is well-known that bile salts solubilize membrane-bound lipids, leading to damage to cell membranes. However, at physiologically relevant concentrations, a simple detergent-like action involving disruption of the membrane is probably not relevant. The intracellular toxicity of bile salts, including death receptor-mediated and/or mitochondria-mediated apoptosis, may play a more important role in inducing the injury of bile duct epithelial cells following liver transplantation. Physiologically, the balance of bile acid influx and efflux in cholangiocytes is maintained by bile acid transporters to avoid the accumulation of toxic bile acids, which is a crucial step of the“cholehepatic shunt,”a process of bile acid absorption by cholangiocytes and subsequent excretion into the peribiliary capillary plexus, followed by resorption by hepatocytes and re-excretion in the form of bile. Several bile acid transporters have been identified in the cholangiocytes in humans and rodents. These transporters are widely expressed on enterocytes, and especially on the terminal ileocytes, where enterohepatic circulation is initiated. Studies show that these transporters will often undergo expressional and functional alterations and may be involved in intestinal metabolic and inflammatory diseases. Based on the above conception and evidence, we hypothesize that due to the expressional and functional alteration of inflammatory factors and nuclear receptor, the cholangiocyte bile acid transport may be disturbed and lost the self-protection from toxic bile, and subsequently inducing the accumulation of toxic bile and bile duct injury.ObjectiveThe current study focuses on the role of cholangiocyte bile acid transporters in bile duct injury after liver transplantation, and the possible mechanism.Methods and results:1. The immunohistochemistry assay on liver tissue section suggested that ASBT and IBABP were expressed exclusively on the large bile duct epithelial cells, while Ostαand Ostβwere expressed on both large and small bile duct. Quantitative PCR and Western blotting analysis showed that the expression levels of Asbt and Ilbp in large bile ducts were significantly higher than those in small bile ducts, while the expression levels of Ostαand Ostβin large bile ducts were similar with those in small bile ducts.2. Using rat liver transplantation model, we investigated the expression levels of cholangiocyte bile acid transporters ASBT, IBABP and Ostα/Ostβ, and evaluated the evaluation of bile duct injury histopathologically. The immunohistochemical assay showed that the expressions of these transporters on large bile ducts were significantly reduced after transplantation compared with the Sham group. Three days after transplantation, ASBT recovered but IBABP, Ostαand Ostβwere still suppressed. Western blot and quantitative PCR analysis showed that the expression levels of these transporters dramatically decreased after transplantation. It took seven to fourteen days for IBABP, Ostαand Ostβto recover, while ASBT recovered within 3 days and even reached a peak above the normal level seven days post operation. In the CP-12h group, the ratios of the ASBT/ILBP, ASBT/Ostαand ASBT/Ostβexpression levels were correlated with the injury severity scores of large but not small bile ducts.3. In the same animal model, we observed the changes of total bile acid (TBA) and phospholipids (PL) concentration in bile, and investigated the expression of FXR by quantitative PCR and Western blottingand, and analyzed its binding ability to promoter of Ostαand Osβgenes by EMSA. Result showed that cold preservation/reperfusion injury is associated with the elevated TBA/PL ratio which could induce the elevation of relative bile acid concentration in bile. It was also showed that the expression of FXR and its binding ability to promoter of target genes were inhibited after liver transplantation, and especially in the transplant group with long donor liver cold preservation time.4. In the in vitro study with cultured rat bile duct units, we observed that the bile with higher TBA/PL ratio, so called toxic bile, could induce the expression of ASBT、IBABP、Ostαand Ostβin normal bile duct. However, after the transfection with FXR shRNA, the expression of ASBT was still induced, while the expression of IBABP、Ostαand Ostβwere inhibited to various extent.Conclusions:1. The bile acid re-absorption may exclusively take places in large bile duct, as the initiator of‘cholehepatic shunt’ASBT is just expressed in large bile ducts.2. The unparallel alteration of cholangiocyte bile acid transporters is significantly correlated with large bile duct injury after liver transplantation with prolonged donor liver preservation.3. The‘toxic bile’could promote the rapid recover and subsequent elevation of ASBT expression, while the IBABP、Ostαand Ostβexpression were not promoted accordingly because of the inhibition of FXR, by which bile acid could induce the expression of IBABP、Ostαand Ostβ.更多还原