肝移植胆道周围血管丛缺血性损伤中的MAC作用机制及对缺血型胆道病变的影响研究

Ischemic type biliary lesion (ITBL) is mainly characterized by the presence of biliary microcirculatory disorders caused by the injury of peribiliary vascular plexus (PVP). Hepatic ischemia reperfusion (IR) is one of significant reason of the ITBL occurrence. Recently, we reported that hepatic IR during the liver transplantation also results in excessive activation of the complement system, which leads to increase of vascular endothelial damage factors. However, inhibition of membrane attack complex (MAC), a terminal product of complement activation, by the complement regulator CD59 improved the pathological lesion. As we known, MAC attacks the cell by changing the intracellular calcium [Ca2+]i. Therefore, we speculate that, the excessive activation of complement system generates MAC during liver IR. MAC combined with endothelia cells of PVP causes a rapid increase in [Ca 2+]i which is dependent on transmembrane channel change, leading to the disorder of bile duct microcirculation， and results in the formation of ITBL. In this project, firstly, we are going to use mCd59ab-/- mouse and rat liver IR model to observe the effect of MAC on PVP; Secondly, to study the injury mechanism of MAC PVP, we will observe the effect of MAC on the induction vascular endothelial cells [Ca2+]i dynamic change in a single cell level; Finally ,we use a transgenic mice, which express human CD59 on its endothelia cells , to define the protect role of human CD59 on the ITBL. The final aim of the study is to get a improved way to prevent ITBL.

缺血型胆道病变(ITBL)主要病理表现为胆管周围血管丛（PVP）损伤所致胆管微循环障碍,内皮细胞是损伤的主要靶点，肝脏缺血再灌注(IR)的是其发生的重要原因。我们已报导，肝脏IR激活补体使血管内皮损伤因子增加，通过CD59抑制补体活化终末产物MAC可改善此病理变化。已知MAC主要通过改变[Ca2+]i浓度攻击细胞。由此推测,肝脏IR可通过激活补体生成MAC,后者与PVP内皮细胞结合,在细胞膜形成跨膜通道改变细胞内外Ca2+浓度使细胞损伤,导致胆管微循环障碍形成ITBL。本研究拟用mCd59ab-/-小鼠肝IR及肝移植模型,观察MAC对PVP损伤作用;在单个细胞水平观测MAC诱导血管内皮细胞[Ca2+]i动态变化，探讨MAC对PVP损伤机制;用内皮细胞表达人类CD59的ThCd59End-/-转基因小鼠,探讨人类CD59对缺血胆道损伤保护作用。最终为改善胆道微循环，预防ITBL提供新思路。

通过以mCd59ab-/-小鼠双基因敲除模型为基础，建立肝脏胆管缺血再灌注模型。并结合大鼠动脉化肝移植模拟肝移植胆管缺血性损伤过程，从补体活化角度，以MAC为切入点，对胆管周围血管丛内皮细胞进行功能学和形态学进行多方面、多层次系统性研究， 初步探讨肝移植及热缺血再灌注过程中MAC对胆管损伤的影响及具体作用机制， 初步揭示了CD59对肝脏缺血再灌注过程中对胆道缺血性损伤的保护作用。 由于补体终末反应产物MAC是肝脏I/RI的重要参与因素之一， 研究结果提示通过CD59抑制补体活化通路MAC形成可减轻在肝癌切除肝门阻断时及肝移植中的肝缺血再灌注补体激活带来的胆管上皮损伤，直接或间接作用于胆管周围血管丛进而引起术后胆管供血不良，多种复合损伤最终导致胆管缺血性损伤， 而干扰这一通路活化将有效减少肝脏缺血再灌注损伤后胆道并发症发生。通过本项目实施，项目负责人获得年国家自然基金项目1项，发表SCI文章4篇，培养博士生1名。综上，项目组基本完成了课题拟定的各项任务，通过本课题的实施，为课题组成员进一步深入研究补体介导肝脏缺血性损伤发生发展机制并探寻临床干预手段和治疗方法提供了坚实基础。