转染血管紧张素转换酶2基因的洋膜间充质干细胞对肺动脉高压引起损伤的血管内皮细胞修复作用及机理

Our previous studies suggest amniotic mesenchymal stem cells (AMMs) are significantly high than other mesenchymal stem cells of differentiation and proliferation of vascular endothelial cells ability. Earlier data exposed that lentivirius transfusion of angiotension converting enzyme 2(ACE2) gene into AMMs resulted in an improved of ACE2 expression and enhanced the capacities of AMMs migration and tube establishment. However, the contribution of ACE2 and AMMs to the development of pulmonary artery hypertension (PAH) is not clear. So we assume, if the transfection of ACE2 on AMMs infusion of PAH, can repair and improve vascular endothelial cell function and reverse pulmonary vascular remodeling, that is an optimization on treatment of PAH. Therefore, we through this project: （1） to investigate whether ACE2 enhances of AMMs differentiation and proliferation of endothelial cells, in turn, whether AMMs can increased the expression and activity of ACE2 in lesions. （2） To investigate molecular mechanism is whether the ACE2 gene by ACE2/Ang1-7/Mas axis, regulating renin angiotensins system balance of through of cellular signal pathway in pulmonary vascular endothelial and smooth muscle cells.（3） By using a standard monocrotaline-induced PAH rat model, were observed the expression levels of ACE2 and its associated downstream molecules will be spotted in the lungs, aiming to clarify the association of ACE2 over-expression and the amelioration of PAH and reveal the possible underlying mechanisms.（4） The expression levels of ACE2 and its associated molecules including Ang1-7 in lung biopsy will be assessed to find the ACE2 with the development and clinical progression of PAH.

我们研究发现，洋膜间充质干细胞（AMMs）对血管内皮细胞分化和增生能力明显强于其它间充质干细胞，其转染血管紧张素转换酶2（ACE2）可增强内皮细胞迁移和成管能力。但转染ACE2的AMMs与肺动脉高压（PAH）疾病关系尚不明确，假设把转染ACE2的AMMs输注PAH，改善血管内皮细胞功能，逆转肺血管重构，使PAH得到优化治疗。通过本课题：（1）研究ACE2是否增强AMMs分化和增生能力，反过来，AMMS是否增加ACE2的表达和活性；（2）通过肺血管内皮细胞及平滑肌细胞的信号通路研究，探讨ACE2基因是否通过ACE2/Ang1-7/Mas轴，逆转肺血管重构的分子机构；（3）通过利用野百合碱诱导的PAH大鼠模型，揭示转染ACE2的AMMs与抗炎，抗增殖的治疗作用机制；（4）检查PAH患者，测定肺组织中ACE2基因及其下游分子含量，揭示ACE2基因与PAH的相关性，为寻找PAH治疗新靶点奠定基础。

我们研究发现，膜间充质干细胞（AMMs）对血管内皮细胞分化和增生能力明显强于其它间充质干细胞，其转染血管紧张素转换酶2（ACE2）可增强内皮细胞迁移和成管能力。但转染ACE2的AMMs与肺动脉高压（PAH）疾病关系尚不明确，假设把转染ACE2的AMMs输注PAH，改善血管内皮细胞功能，逆转肺血管重构，使PAH得到优化治疗。本课题主要研究内容：第一部分细胞水平研究：ACE2-洋膜间充质干细胞制备、ACE2-羊膜间充质干细胞的细胞功能检查，培养脐静脉内皮细胞，加入不同的细胞信号通路抑制剂或激活剂，阐述ACE2/Ang1-7/Mas轴发挥作用可能的分子机制；第二部分为动物水平研究：转染ACE2的人羊膜间充质干细胞对肺动脉高压动物模型治疗作用的研究。研究结果：1、WB检测ACE2-hAMSCs中检测到 ACE2蛋白水平明显升高，P＜0.05。QPCR检测， ACE2-hAMSCs中的血管生成因子及eNOS基因上调，同时下调促炎因子，P＜0.05。2、与PAH组比较，ACE2-hAMSCs组及hAMSCs组的mPAP、RV/LV+S均明显下降， P<0.01，而与hAMSCs组比较，ACE2-hAMSCs组的mPAP、RV/LV+S均有所下降， P<0.01。3、QPCR、WB检测ACE2-hAMSCs中GCP-2、ACE2、Apelin明显升高，而AngⅡ、a-actin明显降低， p＜0.05。4、ELISA检测ACE2-hAMSCs组抗炎因子明显升高的，促炎因子明显降低，P＜0.05。通过本研究表明：ACE2-人羊膜间充质干细胞中 ACE2 的过表达有增强人羊膜间充质干细胞促血管生长因子旁分泌作用。从而致过表达ACE2基因的人羊膜间充质干细胞能有效降低肺动脉高压大鼠模型的肺动脉压力，改善肺血管内皮细胞损伤及右心室重构，其治疗作用优于单纯的羊膜间充质干细胞。ACE2-hAMSCs可能成为肺动脉高压治疗的新策略。