miR506在DCD移植肝缺血性胆管损伤中的作用与机制研究

Ischemic-type biliary lesions(ITBL) is a common disease following liver transplantation (LT) with the donation after cardiac death (DCD), which has an adverse effect on long-term graft survival. Hydrophobic bile salts (HBS) play an important role in the pathogenesis of ITBL. However, the molecular mechanism of this process is still not clear. Recent study showed that HCO3- secreted by cholangiocytes is essential for the protection of biliary epithelial cells (BEC) from HBS cytotoxicity by inhibiting the protonation and subsequent intracellular permeation of HBS. MicroRNA 506 (miR506) is the master regulator of biliary HCO3- secretion. Given the latest report and our preliminary results, we tentatively propose that the up-regulation of miR506 is responsible for the HBS-associated biliary epithelial damage through the decrease of biliary HCO3- secretion and subsequent facilitation of the intracellular permeation of HBS. In this study, we will use the isotopic tracer technique to verify the role of miR506 in biliary HCO3- secretion and bile salt permeation in the cultured BECs and rat DCD LT model. The mechanism of miR506 up-regulation after DCD LT will also be investigated by using gene silencing, luciferase gene reporter assay and other molecular biological tecchniques. In summary, this study will provide new ideas and experimental data for the prevention and treatment of ITBL following DCD LT.

缺血型胆管病变（ITBL）严重影响心脏死亡捐献（DCD）肝移植受者的长期生存。疏水性胆盐（HBS）在ITBL的发生中扮演着关键角色，但具体机制不明。胆管上皮细胞（BEC）所分泌的HCO3-可抑制HBS质子化以阻止其向细胞内渗透，对保护BEC免受HBS损伤具有重要作用。最新研究表明，miR506是BEC分泌HCO3-的关键调控分子。结合前期研究结果，我们提出，肝移植术后miR506表达上调所导致的HCO3-分泌下降可能是诱发HBS细胞内渗透及胆管损伤的主要原因。本研究拟以培养的BEC和大鼠DCD肝移植模型为实验对象，首先利用同位素示踪技术证实miR506表达上调对胆汁HCO3-分泌及HBS细胞内渗透的影响，并分析其与BEC损伤的关系；其次利用RNA干扰、荧光素酶报告基因实验等技术探讨肝移植术后miR506表达上调的分子机制。本研究将为临床上DCD肝移植术后ITBL的防治提供新的思路和证据。

缺血型胆管病变（ITBL）严重影响心脏死亡捐献（DCD）肝移植受者的长期生存。疏水性胆盐（HBS）在ITBL的发生中扮演着关键角色，但具体机制不明。胆管上皮细胞（BEC）所分泌的HCO3-可抑制HBS质子化以阻止其向细胞内渗透，对保护BEC免受HBS损伤具有重要作用。最新研究表明，miR506是BEC分泌HCO3-的关键调控分子。我们推测肝移植术后miR506表达上调所导致的HCO3-分泌下降可能是诱发HBS细胞内渗透及胆管损伤的主要原因。本研究首先利用重建肝动脉血供和胆道外引流的DCD大鼠肝移植模型，深入探讨了缺血缺氧对胆汁HCO3-分泌的影响，揭示了阴离子交换蛋白2（AE2）在这一过程中的关键性作用，明确了胆汁HCO3-屏障在移植肝缺血性胆管损伤发生发展中所扮演的重要角色；其次，通过特异性上调和阻断miR506，观察其对InsP3R3、AE2表达及胆盐渗入量、细胞活性的影响，明确了miR506在缺血缺氧环境下胆汁HCO3-分泌障碍中的作用，提示其可能成为潜在的移植肝缺血性胆管损伤的治疗靶点；最后，探讨了TGR5-STAT3信号通路在miR506表达调控中所扮演的可能角色，发现上调TGR5表达对miR506的表达及功能没有明显的影响，提示缺血缺氧后BEC可能通过其他未知机制进行miR506的调控。本研究加深了我们对移植肝缺血性胆管损伤发病机制的了解，为临床上DCD肝移植术后ITBL的防治提供新的思路和证据。