CD40通路介导炎症微环境对大鼠内皮祖细胞的损伤及其机制的研究

The damaged pulmonary vascular endothelium was repaired through the transplantation of endothelial progenitor cells (EPC). However, inevitably these cells could be impaired in local inflammatory microenvironments of pulmonary vascular lesions. In addition, it was noticed in our previous work (Cytotherapy, 2009, 11) that some of the factors released by EPC had inflammatory effects, which would have EPC impaired. Both the pathway regulating the release of inflammatory mediators by EPC and the route mediating the impairment of EPC by inflammatory mediators are still unclear. In preliminary study, we observed that the expressions of ICAM and IL-6 in EPC membranes were up-regulated through the activation of CD40, which would induce the adhesion and attack of circulating immune cell against EPC itself. Therefore, we are about to carry out the research concerning CD40 pathway mediating EPC impairment in the inflammatory microenvironments and the relative mechanisms to the effects. The contents of this project mainly consist of the following sections. First, in the inflammatory microenvironments imitated in vitro, we are going to investigate the influence of the activation and inactivation of CD40 pathway on the secretion of inflammatory mediators by EPC, and the damage effects of these inflammatory mediators on EPC. And then, key downstream signaling molecules are going to be explored in CD40 pathway, which regulates the secretion of inflammatory mediators by EPC. Lastly, we are going to analyze the role of CD40 pathway in the progression of transplanted EPC impairment caused by the inflammatory microenvironments in vivo. It is anticipated that the pathway mediating EPC impairment in the inflammatory microenvironments will be found, and the theory basis of optimizing cell transplantation will be richened if this research is accomplished.

内皮祖细胞（EPC）移植可修复受损肺血管内皮，但受损肺血管的炎症微环境可影响移植EPC的存活。并且，在前期工作中(Cytotherapy, 2009, 11)，我们发现EPC分泌的部分炎症介质可损伤EPC。然而，EPC分泌炎症介质的调控通路，以及炎症介质损伤EPC的作用途径均尚未清楚。在预实验中，我们观察到CD40激活上调EPC分泌ICAM和IL-6，后两者可粘附免疫细胞并攻击EPC自身。基于此，我们拟深入探讨CD40通路介导炎症微环境对EPC的损伤及其机制，主要内容包括：① 体外模拟炎症微环境并分析CD40通路的激活与失活对EPC分泌炎症介质的影响，以及后者对EPC的损伤效应；② 分析CD40通路调控EPC分泌炎症介质的关键信号分子；③ 在体分析CD40通路在炎症微环境致移植EPC损伤中的作用。本项目的完成将明确炎症微环境致EPC损伤的调控通路，并为细胞移植治疗的优化提供理论基础。

内皮祖细胞（EPCs）是一种特殊的单个核细胞，其在趋化因子的作用下，能迁移至血管损伤及缺血部位，在血管的修复和再生中起重要作用。它们主要通过两种途径发挥生物学功能：分化为成熟内皮细胞和分泌细胞因子。但在多种血管疾病中，EPC的数量和活性均有明显降低。内皮功能障碍在肺动脉高压（PAH）的发生、发展中扮演关键角色，其本质是内皮受损与修复的失衡。越来越多的临床及动物实验数据证明体外扩增的EPC移植对内皮损伤性疾病有治疗效果，其中包括PAH。然而，一些学者提出相反观点。EPC被证明能分泌促炎因子并参与滋养血管的生成。决定移植细胞疗效的关键因素是机体病理微环境。研究证实正常EPC对血管起保护作用，而长期处于疾病环境下的EPC会加重血管损伤。血清中不断增高的可溶性CD40配体（sCD40L）浓度是PAH疾病发展过程中的一个典型病理改变。CD40和sCD40L的联接能通过调控多种免疫及非免疫细胞引发并扩大血管内膜的炎症反应。近年来，CD40通路在PAH疾病发展过程中扮演的角色逐渐受到重视，然而其对移植EPC的调控作用及其潜在机制尚待明确。课题通过在体、离体两个层面的实验，研究了CD40通路对EPC功能的调控作用。主要研究结果包括：⑴ sCD40L浓度梯度损伤EPC体外增殖、迁移、粘附、成血管功能。⑵ sCD40L浓度梯度增加EPC分泌IL-6，ICAM, VEGF的功能。⑶ sCD40L预处理逆转EPC条件培养基对内皮细胞的保护作用。⑷ PAH各项指标于造模两周可见明显改变。各阶段PAH给予EPC移植均有疗效。普通EPC移植效果短暂，shRNA-CD40 EPC疗效更显著且更持久。⑸ shRNA-CD40 EPC较多结合至血管内膜，较少在血管中膜及外膜。从而我们得出结论：sCD40L浓度梯度损伤EPC体外成血管相关功能，但增加其旁分泌功能。PAH模型中，CD40基因沉默的EPC比普通EPC移植能获得更明显且更持久的疗效。综上，我们的研究再次确认了EPC对PAH的治疗作用。EPC单纯移植并不能满足慢性PAH的病变过程，治疗性阻断CD40通路能使EPC更好的发挥治疗效果，是未来EPC移植技术的完善新方向。