内皮祖细胞的免疫调节功能在移植物血管病变中的作用及机制研究

Although major improvements have been made in the prevention and treatment of acute transplant rejection,allograft vasculopathy still limits the long-term survival of patients with heart transplantation.Our previous research indicated that EPCs derived from the recipient were involved in the pathogenesis of allograft vasculopathy and contributed to the neointima formation.However, several issues concerning the contribution of EPCs to the pathogenesis of allograft vasculopathy are controversial. So, it is highly important to clarify the role and the detailed mechanisms of EPCs in allograft vasculopathy. In order to study the immunomodulatory effects fo EPCs,in the present study, we first detect the major histocompatibility complex classⅠandⅡantigen，costimulatory molecules (CD86 and CD80) and CD1d expression by flow cytomerty respectively to evaluate the growth pattern and the phenotype on the surface of EPCs. Meanwhile, we investigate the effects for EPCs on proliferation stimulated by ConA and allogeneic in vitro. The immunoregulatory effect is tested by inhibitory/stimulatory effect on T cell proliferation.Then, we change the number of EPCs from peripheral blood by EPCs mobilization or intravenous transfusion of ex vivo-expanded bone marrow-derived EPCs to evaluate the role of EPCs in the intimal proliferation in a mouse model of allograft vasculopathy.Finally,we investigate the impact of EPCs on immune-related indexes,such as concentrations of several kinds of cytokines and T lymphocyte subsets in peripheral blood and aortic allograft in this mouse model　and study possible mechanisms associated with EPCs and allograft vasculopathy.Based on the understanding to the novel mechanisms of immunoregulatory effects and its mechanism of EPCs to allograft vasculopathy, we prospectively construct a new therapeutic strategy to direct EPCs for the prevention of allograft vasculopathy.

移植物血管病变是导致实体器官移植术后患者死亡的主要中远期并发症之一。我们在前期工作中证实了受体内皮祖细胞（EPCs）参与并促进移植物血管病变，但作用机制仍不清楚。本项目拟对其作用机制进一步深入研究。首先，通过体外检测培养EPCs经诱导分化后细胞表面MHC-Ⅰ、MHC-Ⅱ类抗原与共刺激分子CD80(B-1)、CD86(B7-2)和CD1d分子的表达，观察其可能具有的免疫表型。然后，通过混合淋巴细胞反应（MLC），刀豆蛋白A（ConA）诱导增殖实验以及刺激T淋巴细胞增殖、分化等实验，检测EPCs可能具有的免疫学特性。最后,利用小鼠移植物血管病变模型，通过检测小鼠外周血及移植血管病理标本中EPCs数量，多种免疫相关细胞因子，T淋巴细胞亚型、数量等免疫排斥反应相关指标改变，探讨EPCs在体内对免疫排斥反应的调节作用，阐述EPCs参与移植物血管病变的相关机制，为防治移植物血管提供新的理论依据。

移植物血管病变是导致实体器官移植术后患者死亡的主要中远期并发症之一,内皮祖细胞（EPCs）在血管损伤修复中的发挥重要的作用，但EPCs在移植物血管病变中的作用及机制有待于进一步深入研究。本项目首先利用密度梯度离心法分离小鼠骨髓单个核细胞，并以贴壁培养法定向扩增培养EPCs。然后，EPCs经IFN-γ诱导分化后，检测其细胞表面MHC-Ⅰ、MHC-Ⅱ类抗原与共刺激分子CD80(B-1)、CD86(B7-2)和CD1d等分子的表达情况，结果发现EPCs细胞表面可产生抗原递呈细胞类似的免疫表型。在此基础上，对其进行进一步免疫学功能检测，结果却表明诱导后的EPCs在体外并不能显著刺激T淋巴细胞增殖、分化，也不具备刺激T细胞分泌IFN-γ、IL-10等细胞因子参与相关免疫调节的功能。分析原因可能与EPCs具有的低免疫原性，以及对异体抗原的低反应性有关。同时，利用小鼠移植物血管病变模型，通过自身动员及外源性输入EPCs两条途径，检测EPCs对移植物血管病变的影响。结果表明，EPCs可以促进移植动脉新生内膜形成，但对免疫排斥反应无明显影响。推测EPCs促进移植物血管病变的机制主要与其参与对移植动脉内皮损伤过度修复有关，而非参与或加重受体免疫排斥反应。因此，本项目组根据EPCs的特性，在体外用特异性阻断剂vandetanib阻断EPCs表面受体VEGFR-2活化，发现可以抑制Akt及Erk信号通路，进而明显抑制EPCs的增殖、黏附及迁移等生物学功能。在此基础上，利用小鼠移植物血管病变模型，进一步证实VEGFR-2阻断剂抑制EPCs生物学功能，能有效缓解移植物血管病变的发生。本项目研究为阐明EPCs在移植物血管病变的发病机制中的作用提供新的探索，并为防治移植物血管病变提供新思路。