线粒体自噬功能障碍修复在间充质干细胞减轻高龄供肝缺血再灌注损伤中的机制研究

Hepatic ischemia reperfusion injury (HIRI) is an inevitable pathological process during liver transplantation or other kinds of hepatic surgeries. The critical shortage of liver grafts greatly hampered the progress of liver transplantation. This dilemma is becoming more severe as with the accelerating trend of population aging, so it is of great significance to make full use of liver grafts from aged donors. HIRI fundamentally influences the performance of aged liver grafts and could retard the full recovery of liver function. Our previous study found that aging aggravated HIRI due to decreased mitophagy while increased autophagy could alleviate HIRI. Besides, our published work revealed that mesenchymal stem cells (MSCs) could mitigate HIRI through paracrine effects and mitochondria transfer from MSCs to injured hepatocytes. However, the underlying mechanism remains to be clarified. Based on these previous work, we hypothesize that recovery of dysfunctional mitophagy might be an important pathway accounting for the protective effects of MSCs during HIRI in aged liver grafts. The aim of our present research is trying to delineate the unraveled mechanisms during this process. The cell-cell co-culture system will be used to mimic HIRI in vitro and observe mitochondria transfer from MSCs to hepatocytes as well as the changes of signaling pathways involved in mitophagy with or without MSCs treatment, which will be further confirmed in a model of HIRI in mice of different age group. We will also try to figure out the possible transfer manners, such as tunneling nanotubes (TNTs) and/or microvesicles, which will be adopted by MSCs to deliver healthy mitochondria to injured hepatocytes. Furthermore, we will clarify the key regulatory pathways and factors of how MSCs rescue decreased mitophagy in aged liver grafts. This research will not only elucidate the mechanism of how MSCs ameliorate HIRI from a Mitochondria-Centric perspective, but also facilitate the development of novel strategy for protecting marginal liver grafts based on MSCs application.

随着人口老龄化，有效提高边缘性高龄供肝利用率成为当务之急。高龄供肝相较于正常供肝对肝脏缺血再灌注损伤（HIRI）的耐受性明显降低，易致原发性移植肝无功能。我们研究发现线粒体自噬功能障碍是高龄供肝对HIRI耐受不足的关键，而增加自噬可以减轻HIRI。另外我们前期工作已证实间充质干细胞（MSC）可通过旁分泌和线粒体转运有效减轻HIRI，但更深入的机制仍亟待明确。本课题拟从MSC有效减轻高龄供肝HIRI表象入手，以线粒体自噬功能障碍修复为切入点，通过比较不同年龄组小鼠发生HIRI时及MSC干预治疗前后肝细胞线粒体自噬相关蛋白及信号通路的改变，重点关注和探究MSC对肝细胞线粒体自噬功能修复的主要方式及其分子调控机制。本研究的顺利实施将从细胞器功能修复的微观新视角来阐述MSC减轻高龄供肝HIRI的作用机制，将为有效利用和保护高龄供肝等边缘性供体提供基于MSC治疗的新思路。

本项目总体执行顺利，验证了间充质干细胞（MSC）在减轻高龄供肝缺血再灌注损伤的动物模型中的治疗效果，并着重探讨了MSC发挥保护作用的具体分子机制，取得了与预期目标一致的结果。通过体外细胞实验和小鼠肝脏缺血再灌注损伤（HIRI）模型证实线粒体自噬损伤修复是MSC减轻高龄供肝缺血再灌注损伤的重要作用机制；进一步探讨线粒体的转运方式、修复模式及分子调控机制，从线粒体转运和线粒体自噬障碍修复的新视角来深入阐述 MSC 减轻高龄供肝 HIRI 的作用机制。实验发现在小鼠HIRI模型中，MSCs可上调肝脏细胞内PINK1和Parkin的表达，进而影响细胞内线粒体自噬水平，促进线粒体碎片的消化，减轻功能异常的线粒体在肝脏细胞内堆积，降低线粒体ROS（mtROS）生成，维持线粒体功能，从而改善由于缺血再灌注损伤引起的细胞凋亡。进一步机制研究表明，通过抑制细胞内线粒体自噬或敲除PINK1可显著削弱MSCs的肝脏保护作用，证实MSCs通过修复线粒体自噬损伤减轻高龄供肝缺血再灌注损伤这一过程。本项目的顺利实施为MSCs发挥保护作用的机制提供了新的视角，有助于更好地推进MSCs临床转化应用，增加边缘性供肝利用率提供理论依据，同时也为改造MSC进一步提高其临床疗效及研发基于 MSC 的新型器官保存灌注液提供指导。依托本项目，研究团队在Nature Communications、Cell Death & Disease、Stem Cell Research & Therapy、Molecular Cancer、Genome Biology等杂志上发表相关SCI论文11篇（已标注），申请中国发明专利一项。项目负责人及主要参与人获得后续科研基金7项，培养博士研究生6名，博士后2名。