基于Angiopoietin-1/Tie2信号轴的骨髓间充质干细胞抑制腹主动脉瘤巨噬细胞浸润机制研究

Bone marrow mesenchymal stem cells (MSC) have the potential of anti-immune and anti-inflammation. We have demonstrated that MSC can inhibit the formation of abdominal aortic aneurysm (AAA) through reducing inflammatory cells infiltration and pro-inflammation cytokines expression especially after transfection of angiopoietin-1 (Ang-1) gene, but the mechanism is not yet known. Ang-1 expressed by MSC regulates "non-endothelial cells" such as macrophages is little studied, and whether MSC inhibits AAA formation by the secretion of Ang-1/Tie2 signal is worthy of attention. Therefore, this study intends to clarify the interactions between MSC and macrophages and vascular endothelial cells based on improved AAA model, through in vitro and in vivo experiments using MSC transfected over-expression gene and interfering RNA, Transwell chamber and multi-level molecular biology techniques, to demonstrate the role of Ang-1 in the process of MSC repairing AAA, and to elucidate the mechanism of MSC regulating macrophage infiltration by Ang-1/Tie2 signal axis. This study will reveal the essence of life phenomenon that over-expressed Ang-1 suppresses AAA formation, and provide a novel theoretical basis for applying MSC to repair AAA.

骨髓间充质干细胞（MSC）具抗免疫炎性反应功能，申请者前期研究证实MSC经由减少炎性细胞浸润、下调促炎因子而抑制大鼠腹主动脉瘤（AAA）形成，在转染Angiopoietin-1（Ang-1）基因后尤甚，但机制迄今未明。鉴于MSC表达Ang-1对“非内皮细胞”的巨噬细胞调控作用知之甚少，是否存在MSC通过旁分泌Ang-1/Tie2信号轴抑制AAA形成值得引起关注。因此本课题拟进一步在MSC-巨噬细胞-血管内皮细胞之间Ang-1信号调控角度，基于申请者改进的AAA模型，通过体内外实验，采用慢病毒转染Ang-1过表达和RNA干扰、Transwell小室及多层次分子生物学技术，论证Ang-1在MSC抑制AAA中的作用，阐明MSC经Ang-1/Tie2信号轴调控巨噬细胞浸润的机制。由此揭示Ang-1过表达抑制AAA形成的生命现象本质，为MSC修复AAA的潜在应用前景提供新的理论依据。

骨髓间充质干细胞可以经由减少炎性细胞浸润、下调促炎因子而抑制大鼠腹主动脉瘤（AAA）形成，在转染Ang-1基因后尤甚，但机制迄今未明。我们设想MSC是否通过旁分泌Ang-1/Tie2信号作用于巨噬细胞抑制AAA形成。首先，我们研究了大鼠MSC的生物学特性，将Ang-1过表达和shRNA Ang-1经慢病毒载体包装至MSC形成稳转细胞株，PCR检测MSC-Ang-1具有较高的Ang-1表达水平。其次，我们用裂解外周血的方法获取巨噬细胞，经过流式细胞仪检测，取各组MSC细胞的上清液，通过与内皮细胞共培养、细胞爬片、划痕实验、洗脱实验、Transwell小室等证实，Ang-1促进内皮细胞表达VE-Cadherin粘附分子，抑制巨噬细胞足体运动蛋白F-actin表达。最后，我们将各组MSC细胞回输大鼠AAA模型，体内验证Ang-1及MSC-Ang-1能够通过减少巨噬细胞浸润抑制腹主动脉瘤的形成。另外，我们还在临床实践中通过PET/CT、MLR等方法证实AAA瘤壁和循环中炎症反应状态是其进展及预后的影响因素。这为进一步将MSC用于移植治疗AAA提供理论及实验上的依据。