常温机械灌注抑制DCD供肝分泌CIRP减轻缺血再灌注损伤的机制研究

Normothermic machine perfusion (NMP) can repair DCD liver and relieve ischemia reperfusion injury (IRI). But its mechanism is not clear. We found that a large number of CIRP was accumulated in the DCD liver which under static cold storage, and induced the inflammatory response. But NMP could reduce the secretion of CIRP in the DCD liver. Accordingly, we suppose that NMP can inhibit the secretion of CIRP from DCD liver, reducing Kupffer cells activation and alleviating reperfusion injury. In this study, we plan to compare the expression of CIRP in DCD liver during warm ischemia and cold preservation, and compare the expression of CIRP in primary hepatocytes and Kupffer cells under hypoxia and hypothermia conditions to identify the difference in the phase of CIRP secretion. We will clarify the mechanism of NMP repairing DCD liver by using the liver reperfusion model and liver transplantation model to compare CIRP secretion and IRI of DCD liver under static cold storage, hypothermic oxygenated perfusion and NMP. Moreover, we will explore the possible molecular mechanism of Kupffer cell activation through TLR4 pathway by intervention of recombinant CIRP protein and TLR4 inhibitor in vitro and perfusion of CIRP antibody in vivo. This study not only provides a new target for liver preservation, but also provides a new theoretical basis for the clinical application of NMP.

常温机械灌注（NMP）可修复DCD供肝，减轻缺血再灌注损伤，但具体机制尚未明确。我们发现冷保存人DCD供肝内可蓄积大量CIRP，并诱导炎症反应，而NMP可减少肝内CIRP分泌。据此猜想：NMP可通过抑制供肝离体保存时CIRP分泌，减少再灌注后Kupffer细胞活化，以减轻再灌注损伤。本课题拟通过比较热缺血和冷保存后肝内CIRP的表达、原代肝细胞和Kupffer细胞在低温和缺氧条件下CIRP的表达以明确CIRP分泌的时相差异。利用肝脏再灌注模型和磁吻合肝移植模型比较静态冷保存、低温氧合灌注和NMP对于DCD供肝CIRP分泌及再灌注损伤的影响，阐明NMP修复DCD供肝的作用机制。通过体外重组CIRP蛋白及TLR4抑制剂干预、体内实验灌注CIRP抗体以探索CIRP通过TLR4途径介导Kupffer细胞活化可能的分子机制。本研究不但为供肝保存提供新的干预靶点，也为NMP的临床应用提供新的理论基础。

肝移植是目前治愈终末期肝病的唯一有效手段。大量临床研究证实，常温机械灌注（NMP）能有效减轻移植术后肝脏缺血再灌注损伤，促进肝脏功能恢复。而移植肝离体冷保存期间分泌冷诱导RNA结合蛋白（CIRP），其作为一种损伤相关分子模式分子可触发炎症反应，加重肝脏缺血再灌注损伤。本研究发现冷保存DCD供肝内积蓄大量CIRP，且CIRP含量随冷保存时间延长而增加，并在门静脉开放后释放入血并介导炎症反应，其血清水平在再灌注后2小时达到峰值，是麻醉前的4.5倍，且与术后肝功及IL-6水平正相关。此外，研究明确了DCD肝脏离体冷保存期间主要由Kupffer细胞分泌CIRP蛋白。通过大鼠离体肝脏再灌注模型及rhCIRP蛋白体外细胞干预实验证实分泌型CIRP可通过肝细胞TLR-4途径激活NADPH氧化酶（gp91phox和p47phox），增加肝细胞内ROS释放并介导线粒体动力学失衡，引起肝细胞损伤。与传统静态冷保存及低温氧合灌注方式相比，NMP可通过抑制DCD供肝离体保存期间CIRP的表达分泌，进而减轻后续缺血再灌注损伤，起到修复DCD供肝的作用。在大鼠离体肝脏再灌注模型中，NMP组在肝组织内APT含量、胆汁分泌量、ICG代谢水平、肝细胞内线粒体超微结构、ROS水平、炎症因子水平及细胞凋亡均优于其他两种保存方式；而在低温氧合灌注中加入CIRP竞争性抑制剂C23同样可逆转CIRP介导的缺血再灌注损伤。另外，项目还研发了适用于大动物肝脏及心脏的常温机械灌注设备，可在体外维持肝脏正常代谢吲哚箐绿功能以及维持离体心脏正常搏动和有效输出。本研究的顺利实施不但为DCD供肝保存提供了新的干预靶点，也为常温机械灌注在临床肝移植中的应用提供了新的理论基础。