间充质干细胞对Toll样受体4信号通路负调控减轻肝脏缺血再灌注损伤的机制研究

Hepatic ischemia reperfusion injury (HIRI) fundamentally influences the short-term as well as the long-term performance of liver grafts. Thus, it is crucial to control such inflammatory reactions in order to improve graft function as well as allograft survival. In our previous study, we proved that mesenchymal stem cells (MSC) could alleviate HIRI through inactivation of ERK/MEK pathway. Recent studies showed that the activation of Toll-like receptor (TLR) pathway, especially the activation of TLR4 pathway by endogenous ligands like HMGB1, may be the key trigger to initiate the inflammatory cascade in HIRI. We also found that MSC could suppress DC maturation and function by negatively regulating TLR4 signaling pathway. Based on our previous findings and the important role of TLR4 in HIRI, we hypothesized that the negative regulation of TLR-signaling pathway in hepatic non-parenchyma cells may be the underlying mechanism in which MSCs attenuate HIRI. In our present study, the phenotype and function will be compared in primary liver non-parenchyma cells including Kupffer cells (KC), Dendritic cells (DC), CD4+ T cells, polymorphonuclear cells (PMN) as well as natural killer cells (NK cells), with or without MSC treatment. The expression of TLR, the changes of the downstream and relative pathways will be further studied in the above non-parenchymal cells both in vitro and in vivo. We hope to identify the central players or the key pathway by which MSC negatively regulate TLR- signaling pathway. The results of our present study will provide new insights into the involvement of TLR in anti-inflammatory and immune modulation mechanism of MSC and facilitate its clinical application in liver transplantation.

肝缺血-再灌注损伤(HIRI) 是移植肝功能障碍的重要原因。我们已证实间充质干细胞（MSC）能通过抑制ERK/MEK信号通路来减轻大鼠HIRI（J Surg Res.2012;中华普通外科杂志2011），同时发现MSC可通过抑制Toll样受体4（TLR4）下游信号通路，进而影响DC细胞的成熟和功能（见工作基础）。而TLR4信号通路被新近许多研究证实可能是HIRI中炎症应答最重要的触发器。因此明确MSC是否通过负调控肝间质细胞TLR4信号通路、阻断/减弱后续炎性级联反应来减轻HIRI对于阐明MSC减轻HIRI分子免疫调控机制具有重要研究价值。故本项目从TLRs入手，通过体外体内实验检测多种肝间质细胞被MSC处理前后TLRs表达及其相关信号通路的改变，鉴定出MSC负调控的关键因子或相关负调控信号通路。本研究将从新视角阐明MSC减轻HIRI分子免疫机制，为其在肝移植的转化应用提供理论依据。

肝脏缺血再灌注损伤（hIRI）是移植肝功能障碍的重要原因，但目前仍无有效的治疗手段。间充质干细胞（MSCs）具有免疫调节、抗炎等作用。我们前期研究发现大鼠骨髓间充质干细胞可减轻hIRI，然而其发挥保护作用的分子机制尚未完全清楚。本研究成功分离培养小鼠MSCs并对之进行鉴定；应用MSCs预处理减轻了小鼠70 %部分肝脏缺血再灌注损伤，损伤指标检测发现MSCs预处理组谷丙和谷草转氨酶均降低，肝脏坏死面积及凋亡细胞比例均显著减少，此外MSCs还抑制炎性因子的转录和促进Kupffer细胞由M1型向M2型转变。机制研究发现MSCs可抑制Toll样受体4 (TLR4) /高速率迁移蛋白1 (HMGB1) 蛋白表达，免疫组化及免疫荧光结果均显示MSCs促进HMGB1由肝脏Kupffer细胞核内向细胞质转位，此过程与MSCs抑制Kupffer细胞自噬发生相关。进一步通过酶消化法联合密度梯度离心法将肝细胞和间质细胞分离，再利用免疫磁珠法对CD11b阳性细胞进行阳选，分离原代Kupffer细胞，检测发现TLR4在Kupffer细胞高表达却在肝细胞极少表达。通过建立体外大鼠骨髓MSCs和大鼠Kupffer细胞共培养模型，缺氧 (1%O2) 8小时，复氧6小时，发现MSCs可减少缺氧培养造成的细胞凋亡。对体外模型进行基因表达芯片分析，其结果显示MSCs 减少Kupffer细胞HMGB1的基因表达；通路预测分析预测TLR4与HMGB1之间的中游可能为Akt/mTOR/p-70s6k通路，最后利用实时荧光定量PCR及免疫印迹的方法对基因表达谱预测的Akt/mTOR/p-70s6k通路通路进行了验证。这些结果提示，MSCs抑制小鼠肝脏Kupffer细胞的TLR4/HMGB1通路激活，是其减轻肝脏缺血再灌注损伤的机制之一。本研究初步阐明MSCs减轻hIRI的分子机制，为其在hIRI的转化应用提供新的理论依据，并为hIRI的分子靶向治疗提供新的理论基础。