SDF-1/CXCR7调控内皮祖细胞向脑缺血组织“归巢”的机制研究

It is an important strategy for improvement of treatment efficacy of ischemic cerebrovascular disease by endothelial progenitor cells (EPCs) to enhance its target homing capability. The homing of EPCs is a multistep process regulated by many factors, of which SDF-1 is one of the most important factors. In previous studies, SDF-1 was thought to act through its unique receptor CXCR4. However, SDF-1 was reported to also be a ligand of a novel chemo-kine receptor, CXCR7, which makes people have to study the new mechanism of SDF-1.In our previous researches, we have found that CXCR7 is not only highly expressed in EPCs and also involves in regulating EPCs adhesion, transendothelial-migration behvaviors, which suggest that CXCR7 should be involved in regulating the homing capability of EPCs, but its mechanism is unclear. In this proposal, we will establish a multi-parameter controlled angiogenesis model based on microfluidic technology and combine with acute cerebral ischemia model to demonstrate the functions of SDF-1/CXCR7 in the homing of EPCs to cerebral ischemia tissue by RNAi technology and fluorescent tracer technique etc. And we will further clarify the key signaling pathways of SDF-1/CXCR7 involved in EPCs homing based on its functions. In summary, we hope this proposal could provide a new target for enhancing EPCs homing to sites of cerebral ischemic and provide a new strategy for the clinical application of of stem cell therapy for ischemic diseases.

增加内皮祖细胞（EPCs）的靶向"归巢"是提高EPCs治疗缺血性脑血管病疗效的重要策略。EPCs的"归巢"是受多因素调节的多步骤过程，其中SDF-1是最受关注的因子之一，但既往研究都认为SDF-1通过其唯一受体CXCR4发挥作用，然而，新受体CXCR7的发现使得人们需要研究SDF-1新的作用机制。我们前期研究发现CXCR7不仅在EPCs中高表达，并对EPCs粘附、跨内膜转移等行为发挥着重要作用，提示CXCR7应该参与EPCs"归巢"能力的调节，但其作用机制尚不明确。本项目将基于微流控技术构建多参数可控的血管生成模型并结合大鼠急性脑缺血模型，利用RNAi技术、荧光示踪技术等解析SDF-1/CXCR7在EPCs靶向脑缺血组织"归巢"这一复杂过程中的作用，并将进一步阐明其关键作用信号途径，以期为促进EPCs向脑缺血组织"归巢"提供新的作用靶点，并为干细胞治疗缺血性疾病的临床应用提供新策略。

缺血性疾病中通常伴随内皮祖细胞(Endothelial progenitor cells，EPCs)数量的减少，功能的受损，如何增加EPCs数量，提高EPCs靶向“归巢”到缺血部位并促进其参与血管新生是治疗缺血疾病的重要的策略之一。我们前期研究发现CXCR7在EPCs的存活和血管新生中发挥着重要的作用，本项目拟考察上调CXCR7表达对EPCs血管新生功能的改善作用及其分子机制。首先我们通过构建CXCR7重组慢病毒载体，转染骨髓小鼠来源的EPCs，成功构建高表达CXCR7的EPCs细胞（Lv-CXCR7-EPCs），同时对照细胞为只转染含有GFP的对照载体（Lv-null-EPCs）。我们通过体外和体内模型考察上调CXCR7的表达对EPCs功能的影响。我们利用ox-LDL模拟体内的高脂条件处理EPCs，结果发现ox-LDL诱导EPCs凋亡，抑制EPCs管样结构的形成，阻止EPCs的跨内皮迁移；而上调EPC中CXCR7的表达，加入其配体SDF-1在一定程度上能阻止EPCs的凋亡，增加EPCs的跨内皮迁移能力且能改善EPCs的血管形成能力。我们进一步利用大鼠脑缺血模型和下肢缺血模型评价上调CXCR7的表达对EPCs“归巢”和参与血管新生中的作用，结果发现上调CXCR7的表达能够促进EPC“归巢”到缺血部位，增加EPC整合到毛细血管中，并且能够通过分泌促血管生长因子VEGF促进微循环的重构。 .我们进一步探索了上调CXCR7改善EPCs血管新生能力的作用机制。氧化应激的增加是缺血性疾病中EPCs功能受损的重要机制，ox-LDL处理显著提高细胞内活性氧（ROS）含量，而上调CXCR7的表达能够显著降低ROS的升高。转录因子Nrf2是机体抗氧化损伤的重要调控因子。我们发现上调CXCR7的表达可以提高Nrf2在细胞核内的分布，增强其下游抗氧化基因NQO-1，HO-1及Catalase的表达。随后，我们进一步研究了CXCR7调控Nrf2在细胞核内停留的分子机制。结果发现上调CXCR7是通过激活Akt-GSK3β-Fyn信号通路调控Nrf2转录活性。.综上所述，该项目证实了上调CXCR7能够增加EPCs的靶向“归巢”能力，同时还能改善其在缺血性疾病中的血管新生的能力，并揭示其新的作用机制，为EPCs治疗缺血性疾病提供了新的基因修饰靶点。