间充质干细胞来源的miR-106b-25基因簇调控FoxO3a信号通路减轻移植肝缺血再灌注损伤的作用及机制研究

Hepatic ischemia-reperfusion injury (HIRI) is an important cause of liver graft dysfunction. Stem cell therapy shows good prospects. Thus, we believe that it is helpful to significantly improve the therapeutic effect of stem cells through exploring the underlying mechanism of liver graft injury and taking appropriate intervention measures. We have confirmed that the nuclear transcription factor FoxO3a affected liver transplantation-induced organ damage through regulating apoptosis and DNA repair (National Natural Science Foundation of China, No.81571926). We analyzed the results of the gene chip and found that the miR-106b-25 gene cluster derived from mesenchymal stem cells (MSCs) might play an important role in the protection of liver graft. Bioinformatics combined with pretest results showed that miR-106b-25 gene clusters could be transferred to hepatocytes through gap junction or exosomes, and affect the activity of FoxO3a through regulating PI3K/Akt and HIC1/Sirt1 signaling pathways. Therefore, we speculate that the miR-106b-25 gene cluster derived from MSCs can protect the liver graft through regulating FoxO3a. This project is based on our previous studies, which explores the way of miR-106b-25 gene cluster transferring to hepatocytes from MSCs, the mechanism of miR-106b-25 regulating the activity of FoxO3a, and the role of miR-106b-25 in the protection of liver graft, so as to find out a new mechanism of MSCs protecting against HIRI of liver graft, providing new theoretical basis for its clinical transformation and application.

肝缺血再灌注损伤（HIRI）是移植肝功能障碍的重要原因，干细胞治疗展现良好前景，明确主要机制并行干预可望显著提高干细胞的治疗效果。我们已证实核转录因子FoxO3a通过调控凋亡及DNA修复而减轻器官损伤（前国自项目）；采用基因芯片发现源于间充质干细胞（MSCs）的miR-106b-25基因簇在移植肝保护中可能发挥重要作用；生物信息学结合预实验结果显示该基因簇可以通过缝隙连接或外泌体传递到肝细胞，通过调节PI3K/Akt和HIC1/Sirt1信号通路而影响FoxO3a的活性。因此推测源于MSCs的miR-106b-25基因簇通过调控FoxO3a对移植肝产生保护作用。本项目拟在前期工作基础上，通过体内外实验探讨miR-106b-25基因簇从MSCs传递至肝细胞的途径、对FoxO3a活性的调控以及在移植肝保护中的作用等，以期发现MSCs减轻移植肝HIRI的新机制，为其临床转化应用提供新的理论依据。

移植肝损伤是肝移植研究领域面临的重要难题，严重的影响了肝移植患者的术后生存率。在本项目研究过程中，我们发现和证实 Cx32 蛋白的表达和功能在移植肝缺血再灌注损伤中起关键作用。首先，我们发现了Cx32蛋白表达水平和移植肝损伤的严重程度呈正相关关系，尤其在移植肝再灌注6小时后，肝损伤最严重伴随Cx32蛋白表达水平达到峰值。并且，Cx32蛋白可以直接激活 PKCα，随后激活了促炎相关的 NF-κB/NLRP3 信号通路，诱导肝细胞大量生成 ROS ，后者激活了凋亡相关蛋白（Bak/Bax、Cyto C和Caspase 3），引发肝细胞凋亡，最终导致移植肝损伤；重要的是，大量生成的 ROS 还可以通过由 Cx32 蛋白组成的缝隙连接快速地传递到邻近的肝细胞内，同样激活凋亡相关蛋白引发细胞凋亡，最终加重移植肝损伤。而且，通过人骨髓间充质干细胞预处理，我们发现干细胞可以显著减轻移植肝损伤；其中，人骨髓间充质干细胞不仅通过与肝细胞上的Cx32蛋白形成缝隙连接直接作用于肝细胞，还可以通过释放外泌体间接作用于肝细胞，从而减轻移植肝损伤。通过本项目的研究，我们发现了介导移植肝缺血再灌注损伤的新机制及潜在的治疗新策略，为移植肝的保护提供了一系列可能的治疗靶点，也为药物的开发初步打下了基础。