骨髓间充质干细胞对急性肝衰竭Treg/Th17细胞失衡的调节及腺苷A2A受体的免疫介导机制

The imbalance of immune cells such as Treg (anti-inflammatory) /Th17 (pro-inflammatory) cells and the inflammatory cascade play a leading role in the progression of acute liver failure (ALF). The previous works showed that bone marrow mesenchymal stem cells (BMMSCs) have the roles of negative immune regulation and anti-inflammatory effects in end-stage liver disease, relating to the correction of Treg/Th17 imbalance. But the mechanisms are still unclear. Accumulating evidences suggested that the activation of adenosine A2A receptor (A2AR) on the surface of immune cells is crucial for the immunoregulation of anti-inflammatory and negative immune effects. A2AR is also closely related to the differentiation and the function of Treg cells. According to above results, we propose a hypothesis that BMMSCs under the inflammatory environment of ALF may correct the imbalance of Treg/Th17 cells via activing downstream signaling pathways of A2AR which on the surface of T cells. To address this, the antagonists and agonists related to A2AR signaling pathways, and the A2AR-KO mice will be used in the present study by the methods of co-culture in vitro and transplantation in vivo to clarify the immune regulation effects of BMMSCs on the T cells such as the imbalance of Treg/Th17 cells in ALF, and further to understand the immune-mediated mechanism of A2AR. This study will provide an important theoretical basis for the transplantation of BMMSCs in ALF, and also provide a new therapeutic target for the immune reconstruction of ALF.

急性肝衰竭（ALF）病程进展中免疫细胞如Treg（抑炎）/Th17（促炎）失衡及炎症瀑布样反应起着主导作用。申请人前期工作表明骨髓间充质干细胞（BMMSCs）对终末期肝病具有负向免疫调控及抑炎作用，并纠正Treg/Th17细胞失衡，但具体机制仍不清楚。而免疫细胞表面腺苷A2A受体（A2AR）在抗炎和负向免疫调控方面至关重要，并与诱导Treg细胞分化及功能改变密切相关。因此，本项目提出“ALF炎性微环境下BMMSCs可通过T细胞表面A2AR，激活其下游信号通路而调节Treg/Th17细胞失衡”这一假说。为验证该假设，本项目使用相应拮抗剂和激动剂及A2AR基因敲除小鼠，通过体外共培养和体内移植治疗等方法探讨BMMSCs对T细胞免疫如Treg/Th17细胞失衡的调控作用，进一步明确A2AR的免疫介导机制，为BMMSCs治疗ALF提供重要的理论依据，同时也为ALF的免疫重建提供新靶点。

各种病因引起的急性肝衰竭预后差，治疗手段有限，严重危害着人类健康。大量文献表明急性肝衰竭病程中免疫细胞的失衡如Treg/Th17细胞及炎症瀑布样反应起着关键作用，如何调节失衡的免疫细胞，阻断炎症细胞因子风暴，促进肝细胞再生修复是广大学者研究的重点和难点问题。本课题组建立急性肝衰竭模型，成功分离、培养及鉴定骨髓间充质干细胞（BMMSCs），并将BMMSCs经鼠尾静脉移植，发现BMMSCs移植可显著上调Treg细胞比例，抑制肝内炎症；体外 HSC 与 BMMSCs 共培养发现可通过腺苷 A2A 受体相关的腺苷-CD39-CD73 信号通路降低 HSC 激活、炎症细胞因子和胶原蛋白产生，发挥其抗肝损伤和肝纤维化作用；而腺苷A2AR激动剂联合BMMSCs移植则更起到协同调节作用，进一步抑制TLR4/NF-κB通路，显著上调ALF小鼠体内Treg细胞比例。研究提示BMMSCs移植治疗急性肝衰竭可能与腺苷/腺苷A2AR通路以及免疫细胞的调节密切相关，并可能通过干扰LPS/TLR4/NF-κB信号通路抑制肝内炎症，负向免疫调节，从而改善急性肝衰竭预后。另外本项目组通过临床研究，发现BMMSCs在不同的微环境下，呈现出不同的分泌和免疫调节功能，BMMSCs 对肝衰竭来源的 CD4+ T 细胞存在负向免疫调节作用。而正常人却没有类似调节作用。 BMMSCs 在两组中免疫效应的差异可能与血清中所含刺激因子的不同相关。本项目经过上述一系列动物和临床研究，目前已经发表高质量SCI、中华系列及核心期刊共6篇，相关成果获浙江省科技进步奖二等奖1项，申报专利1项（实质审查中），进一步优化了临床干细胞移植技术，培养硕士3人，为骨髓干细胞移植治疗终末期肝病提供了重要的理论和实践依据，具有非常重大的临床应用价值和社会意义。