人Tim-1在小鼠肝脏缺血再灌注损伤及肝再生中的作用与分子机制研究

Recent studies have found that T cells were involved in the occurrence and development of HIRI, human Tim-1 could bind to and inhibit the reactivity of human and mouse T cells. In our previous work, we synthesized the humanized Tim-1-Fc and found it could inhibit the activation and proliferation of mouse CD4+ T cell, inhibit the Th1 response, and extend the survival of heart allograft. However, the value of human Tim-1 in HIRI and liver regeneration in mice remains unclear. This study intends to adopt mice HIRI and partial hepatectomy model to detect the level of postoperative serum transaminases and liver histopathological changes, the infiltration of inflammatory cells in the liver, the apoptosis and regeneration of liver cells, so as to analyse the role of Tim-1-Fc in mouse HIRI and liver regeneration. In addition, we intent to detect the level of liver tissue oxidative stress, inflammatory factors, adhesion molecules, membrane surface molecules and their endogenous ligands, growth factors and intracellular expression and activation of signaling molecules, so as to reveal the value and molecular mechanisms of Tim-1-Fc in alleviating HIRI and promoting liver regeneration by the application of a variety of cellular immunology and molecular biology methods. This study is expected to provide scientific basis for the usage of Tim-1-Fc as an immunosuppressive agents in clinical HIRI and liver transplant anti-rejection treatment, which has important theoretical significance and practical value.

近年来研究发现T细胞参与了HIRI的发生发展，人Tim-1可与人和小鼠T细胞结合并抑制其反应性。我们前期工作合成了人源化Tim-1-Fc，发现其可抑制小鼠CD4+T细胞活化增殖，抑制Th1反应，延长同种心脏移植物存活期。但人Tim-1在小鼠HIRI及肝再生中的作用仍不清楚。本课题拟采用小鼠HIRI及部分肝切除模型，通过检测术后血清转氨酶及肝组织病理学改变，观察肝脏内炎性细胞浸润，肝细胞凋亡和再生情况，分析Tim-1-Fc在HIRI及肝再生中的作用；并应用多种细胞免疫学和分子生物学方法检测肝组织氧化应激水平，炎性因子、黏附分子、膜表面分子及其内源性配体、生长因子和胞内信号分子的表达与活化情况，揭示Tim-1-Fc减轻HIRI，促进肝再生的分子机制。项目研究结果有望为Tim-1-Fc作为一种新型免疫抑制剂应用于临床HIRI及肝移植抗排斥治疗提供科学依据。具有重要的理论意义和实用价值。

背景：本研究在前期研究发现Tim-1-Fc具有免疫抑制作用，能显著减轻小鼠心脏移植物排斥反应的基础上。探讨其在小鼠缺血再灌注损伤及肝脏再生中的作用。内容：我们采用了小鼠70%肝脏缺血再灌注损伤（HIRI）模型和HIRI+50%肝切除模型，收集小鼠再灌注后不同时间点的血液和肝组织标本，通过酶学检测、病理和免疫组化、ELISA、PCR、Western-blot，组织氧化和细胞周期分析等方法，多方面评判Tim-1-Fc对小鼠HIRI和肝脏再生的影响。结果：Tim-1-Fc能通过减少肝内炎性（巨噬，T细胞和中性粒）细胞浸润，抑制小鼠体内炎性细胞因子（TNF-α、IL-1β、IL-6、IFN-γ、MCP-1、MIP-2）、粘附分子（VAP-1、ICAM-1、VCAM-1）、膜表面分子（CD40、TLR4）及其内源性配体（HMGB1、HSP70）表达，调节肝组织中氧化（MPO，MDA）/抗氧化（SOD）平衡，抑制MAPK信号途径分子（ERK，JNK，P38，P65）磷酸化等机制，显著减轻小鼠HIRI（酶学水平和病理检查）；并通过抑制炎性因子IL-2表达，调节肝组织中氧化/抗氧化平衡，增加生长因子（IGF-1，HGF）表达，增强PI3K/AKT信号途径等机制促进肝再生。意义：本研究发现Tim-1-Fc作为一种免疫抑制剂可显著减轻小鼠HIRI，促进肝再生。为其临床应用提供了实验基础。