移植肝脏组织内Foxp3+/IL-17＋表达模式诱导急性排斥反应的机制研究

The application subject is based on our previous founding that the numbers of expressing Foxp3+ or IL-17+ cells on the allograft tissue increase obviously with acute rejection in liver transplant patients, and the phenotype and function of Treg and Th17 cells are associated with the injury of liver function. From the previous discovery, we speculate that allograft environment would improve part of Foxp3+ cells convert to the Fxop3+IL-17+ double positive cells, then increase acute rejection and affect the progress of liver disease. This subject will analysis the expression pattern of Foxp3+ and IL-17+ in liver tissue, demonstrate the mechanism of liver tissue microenvironment regulation on Foxp3+/IL-17+ expression and induction the impairment for allograft. Also, we analysis the regular patterns of frequency and function of Treg and Th17 cells in disease progress after liver transplantation. Furthermore, we will illustrate the possibility of Treg convert to the Th17 cells and regulation of Treg on Th17 cells in vitro. Finally, we will establish the liver transplant animal model to demonstrate the feasibility of using Treg and anti-IL-17 for prevention and treatment of rejection. The application subject will not only help understanding the imbalance of Treg/Th17 in liver tissue, demonstrating the immunological mechanism of acute rejection, but also to find the immunological indexes for aided diagnosis of acute rejection, and novel strategy for treatment of rejection.

本课题基于申请者前期发现术后急性排斥者移植肝组织内表达Foxp3+和IL-17+细胞均增加，Treg和Th17的表型和功能与肝脏损伤之间存在相关性，推测移植肝内微环境可能促进部分Foxp3+细胞转化为Foxp3+IL-17+细胞，加重排斥反应，影响疾病的进展。本课题拟进一步分析肝组织内Foxp3+和IL-17+表达模式，探讨肝脏微环境调控Foxp3+/IL-17+表达导致肝脏损伤的机制，同时观察肝移植术后疾病自然进程中Treg和Th17频率和功能的变化规律。体外观察肝移植中Treg转化为Th17以及对Th17的调节作用。建立肝移植急性排斥动物模型，研究利用Treg和anti-IL-17防治排斥反应的可行性。本研究不但有助于理解肝组织内Treg/Th17失衡，阐明肝移植急性排斥反应的免疫学机制，而且有可能发现辅助诊断肝移植术后急性排斥反应的免疫学指标，以及治疗排斥反应发生的新方法。

肝移植术后发生急性排斥反应的机制尚未完全阐明，目前认为调节性T细胞（Treg）、Th17细胞参与了移植术后的免疫耐受和免疫排斥反应。近来发现滤泡辅助性T细胞（Tfh）和滤泡杀伤性T细胞（Tfc）也参与了小肠移植、肾移植、造血干细胞移植等免疫反应，但在肝移植免疫中的作用及特点尚不清楚。为此，本课题将首次探讨肝移植患者Tfh细胞和Tfc细胞表达特点，结合临床资料，探讨Tfh、Tfc细胞参与排斥反应的可能机制。. 研究取得如下成果：（1）术后4周Tfh2细胞频率较移植前明显升高，而Tfc细胞在发生急性排斥反应的患者外周血中明显高于非排斥组和健康对照组。（2）移植术后总B细胞和幼稚B细胞频率非排斥组和排斥组均高于健康对照组，而记忆B细胞和浆细胞频率健康对照组高于非排斥组和排斥组。（3）外周血Tfc细胞频率与总B细胞和幼稚B细胞频率均具有明显的正相关性，与记忆B细胞和浆细胞有明显的负相关性。（4）患者外周血中Tfc细胞频率与谷氨酸转移酶（ALT）和天冬氨酸转移酶（AST）水平没有明显的相关性，但与谷氨酰转移酶（GGT）水平呈明显的正相关关系。（5）脐带充质干细胞（UC-MSC）对肝移植术后发生的急性排斥反应有减轻肝炎炎症的作用，能明显提升患者外周血中Treg细胞以及血清中TGF-β1和PGE2水平，降低Th17细胞的水平，下调CD4T细胞的活化。. 总之，本课题首次探讨了Tfh细胞和Tfc细胞在肝移植中的表达特征及其意义，提出Tfh和Tfc可能参与了肝移植术后的移植物免疫反应。同时探讨了UC-MSC治疗移植肝急性排斥反应及其相关机制，具有重要的理论和现实意义。