PD-1调控糖酵解介导慢加急性肝衰竭T淋巴细胞功能障碍的机制研究

Patients with acute-on-chronic liver failure (ACLF) at middle and advanced stage are often in a state of persistent immunosuppression, which can lead to secondary infection and associated complications, thus increasing mortality. T cell dysfunction is an important part of ACLF immunosuppression, but its specific mechanism remains unclear. Energy metabolism is a basic factor to regulate cell function, and glycolysis is a predominantenergy delivering pathway for T cell activation.We found in our earlier studies that the level of glycolysis in T cells was decreased, the immune function was inhibited, and the function of T cells was affected by abnormal glycolysis in ACLF patients. Programmed cell death-1 (PD-1) negatively regulated T cell functions. In addition, we found that blocking PD-1 could improve the glycolysis and the function of T cells in ACLF rats. Thus, we proposed the hypothesis that PD-1 regulates T lymphocyte dysfunction by glycolytic pathway in ACLF. In our project, we will investigate the relationship between T cell function and glycolysis abnormality in ACLF in various aspects, including clinical patients, in vitro cells and animal models, and explore the role of T cell glycolysis and mTOR signaling pathway in the regulation of PD-1 in ACLF. This study aims to explore the role of PD-1 and glycolysis in the development of ACLF immunosuppression and the associated molecular mechanisms based on a new perspective of cell energy metabolism in order to provide a novel theoretical evidence for the prevention and treatment of diseases.

慢加急性肝衰竭（ACLF）患者中晚期常处于持续的免疫抑制状态，易引发继发感染及相关并发症，增加死亡率。T细胞功能障碍是ACLF免疫抑制重要部分，但其具体机制尚不明确。能量代谢是调控细胞功能的基本因素，其中糖酵解是T 细胞活化最主要供能方式。我们前期发现，ACLF患者T细胞糖酵解水平下降，免疫功能抑制，糖酵解异常影响T细胞功能。程序性死亡分子1(PD-1)负性调控T细胞功能。我们进一步发现阻断PD-1可改善ACLF大鼠糖酵解水平，改善T细胞功能。由此提出研究假说：PD-1调控细胞糖酵解途径介导ACLF时T细胞功能障碍。本项目将从临床患者、离体细胞和动物模型多层面，探索ACLF时T细胞功能与糖酵解异常的关系；探索PD-1调控ACLF中T细胞糖酵解作用方式及信号通路。本研究从细胞能量代谢这一新角度，探索PD-1及糖酵解在ACLF免疫抑制过程中的作用及相关分子机制，为疾病防治提供新的理论依据。

目的：探索PD-1是否参与乙型肝炎相关的慢加急性肝衰竭(HBV-ACLF)患者外周血CD8+T淋巴细胞功能障碍。.方法：收集HBV-ACLF患者和健康对照者外周血，获得外周血单个核细胞（PBMC），流式细胞术检测CD8+T淋巴细胞的数量及CD8+T淋巴细胞PD-1的表达状况。进一步体外培养ACLF患者的外周血分选的CD8+T细胞，一组加入PD-L1-IgG融合蛋白（ACLF+PD-1组），一组加入IgG融合蛋白（ACLF组），分析其增殖能力（ki67）和细胞活力（CD69）及分泌效应细胞因子（IL-2、IFN-γ、TNF-α）能力。.结果：入组的30例HBV-ACLF患者和健康对照者，ACLF患者外周血CD8+T细胞绝对数（333.88±147.74）明显低于健康对照者（872.50±206.64）（F=2.760，P<0.001）。相对于健康者（7.02±2.12%），ACLF患者外周血的CD8+ T 淋巴细胞PD-1表达明显升高（13.33±2.52%）,（F=0.203，P=0.027）。在体外培养，相对于健康者，ACLF患者外周血CD8+T细胞活力（CD69）和增殖能力（ki67）均减弱（均P＜0.001）；产生IL-2、IFN-γ、TNF-α水平减弱（均P＜0.001）。相对于ACLF组，ACLF+ PD-1组CD8+T细胞其增殖能力（ki67）和细胞活力（CD69）进一步减弱（均P＜0.05）；产生IL-2、IFN-γ、TNF-α水平进一步降低（均P＜0.05）。.结论：HBV-ACLF患者存在CD8+T淋巴细胞功能障碍，PD-1可能参与调控ACLF患者的CD8+T淋巴细胞功能障碍。