遗传谱系示踪技术研究c-kit+干/祖细胞参与血管损伤修复及内膜增生的作用及病理机制

An increasing number of studies suggested that stem/progenitor cells may differentiate to endothelial cells (ECs), vascualr smooth muscle cells (VSMCs) or immuno-inflammatory cells in vascular diseases. However, there is no direct and overwhelming evidence demonstrating the differentiation fate of stem/progenitor cells under pathophysiological conditions of vascular injury-repair and remodeling, due to the limitation of methodology. Genetic lineage tracing, which is a new important tool to study stem cell differentiation, can trace the cell origin and fate in vivo. We have generated Kit-CreER;Rosa26-RFP mice that are capable of labeling endogenous c-kit+ stem/progenitor cells after tamoxifen administration in vivo. In this project, by using Kit-CreER;Rosa26-RFP mice, we will perform mouse femoral artery wire injury-induced neointima formation model and carotid artery ligation-induced neointimal hyperplasia model, investigating whether endogenous c-kit+ stem/progenitor cells can differentiate to ECs, VSMCs or immuno-inflammatory cells to repair or remodel the injuried arteries in vivo. Furthermore, whether bone marrow-derived or perivascular c-kit+ stem/progenitor cells contribute to vascular injury-induced neointimal hyperplasia will be studied via bone marrow transplantation with/without perivascular tissue removal. Finally, ACK2 (c-kit receptor blocking antibody) will be administered intravenously to deplete endogenous c-kit+ stem/progenitor cells, consequently evaluating their roles in vascular neointima formation. This project will provide strong and convincing evidence showing whether stem/progenitor cells participate in vascular injury-repair response and remodeling process, and uncovering the effect of c-kit+ stem/progenitor cells in vascular injury induced neointimal hyperplasia.

大量研究提示干/祖细胞可能分化为ECs、VSMCs和免疫炎症细胞参与血管疾病，然而局限于研究技术方法，目前仍缺乏体内病理生理状态下干/祖细胞分化参与血管损伤修复和重塑的直接证据。遗传谱系示踪技术可在体内追踪细胞来源和转分化命运，是研究干细胞分化的关键新工具。本项目利用可标记c-kit+干/祖细胞的Kit-CreER;Rosa26-RFP谱系示踪小鼠，行股动脉导丝损伤和颈动脉结扎内膜增生模型，研究c-kit+干/祖细胞能否分化为ECs、VSMCs或者免疫炎症细胞参与血管损伤后内膜增生；利用骨髓移植和/或血管外周组织剥除实验探究到底是骨髓来源还是血管外周来源的c-kit+干/祖细胞参与了血管重构；利用ACK2（c-kit受体封闭抗体）消除体内c-kit+干/祖细胞，研究其是否影响血管损伤后的内膜增生程度。通过本项目研究，可为c-kit+干/祖细胞如何参与血管损伤内膜增生提供直接证据。

背景：越来越多证据表明血管干细胞参与血管损伤后修复及重构过程，但仍存在众多争议，一个核心的关键问题是：干细胞到底能不能在体内病理生理状态下直接分化成血管相关细胞参与血管损伤修复及重塑？c-kit+干细胞曾被报道在体外培养及缺血组织中可分化为内皮细胞及平滑肌细胞。在本研究中，我们利用新技术新方法研究内源性的c-kit+干细胞是否及如何参与血管损伤后内膜增生病变。.研究方法及结果：我们构建了可在体示踪c-kit+干细胞的Kit-CreERxRosa26-RFP谱系示踪小鼠。利用谱系示踪技术，我们发现血管干细胞的作用非常复杂，与既往的认知不同，在血管损伤病理性重构过程中，c-kit+干细胞并不分化成内皮细胞和平滑肌细胞，而是转分化成免疫炎症细胞参与血管损伤后炎症反应。尽管c-kit+干细胞存在于骨髓、血管壁中，但谱系示踪结合骨髓移植结果表明，骨髓来源c-kit+ 干细胞是促进内膜增生的主要来源。利用c-kit+干细胞的阻断抗体ACK2抑制c-kit+干细胞的转分化有利于减轻血管病理性重构。.结论及科学意义：c-kit+干细胞在内皮损伤后血管重构过程中并不修复血管，而是参与炎症反应加重血管病变，应用c-kit+干细胞阻断抗体ACK2可能有助于减轻血管损伤后内膜增生导致的管腔狭窄，为心血管介入术后再狭窄提供了新的干预思路。