【作者】 李敏才；

【导师】 阮秋蓉；

【作者基本信息】 华中科技大学 ， 病理学与病理生理学， 2010， 博士

【摘要】 目的：骨髓间充质干细胞(bone mesenchymal stem cells, BMSCs)对以细胞为基础的干细胞治疗、特别是对于心血管疾病如心肌梗死、动脉粥样硬化等治疗,具有非常大的吸引力和应用前景。然而BMSCs是如何趋化、迁移到病变部位的分子机制并不十分清楚。目前已知基质细胞衍生因子1 (stromal cell-derived factor-1, SDF-1)是影响干/祖细胞迁移的关键趋化因子之一,并参与动脉粥样硬化过程。本实验旨在研究内皮细胞分泌的SDF-1是否影响CXCR4+干/祖细胞向内皮损伤处迁移与粘附。方法：首先从动物骨髓分离并培养BMSCs,通过磁珠分选出CXCR4+BMSCs;用氧化低密度脂蛋白(ox-LDL)刺激人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)诱导SDF-1 a表达；通过RT-PCR和ELISA方法检测HUVECs中SDF-lαmRNA和蛋白表达。采用Transwell小室检测CXCR4+BMSCs迁移功能；最后检测HUVECs分泌上清中SDF-1α对CXCR4+BMSCs粘附影响。结果：研究发现在ox-LDL刺激下,HUVECs中SDF-1αmRNA和蛋白升高。同时HUVECs低氧诱导因子(hypoxia-inducible factor, HIF-lα)表达上调。与对照组相比,含SDF-1 a上清促进了CXCR4+BMSCs迁移；这种迁移现象可被预先孵育CXCR4抗体抑制。与对照组相比,CXCR4+BMSCs粘附功能显著增强；而CXCR4中和性抗体可抑制这种粘附现象。结论：我们的结果提示在致动脉粥样硬化(atherosclerosis, AS)病变过程中,ox-LDL刺激HUVECs通过SDF-1α/CXCR4轴引起CXCR4+BMSCs发生迁移与粘附运动。目的：近来研究发现骨髓间充质干细胞(BMSCs)参与了损伤后修复过程中新生血管形成和缺血梗死后的修复。骨髓源性CXCR4+干/祖细胞经动员到梗死区域促进梗死修复,然而并不明确干/祖细胞对缺血梗死后修复的作用机制。本课题在体内、体外研究分析CXCR4+BMSCs的血管形成能力,探讨其对缺血损伤后的修复机制。方法：通过磁珠分选并培养CXCR4+BMSCs,用血管内皮生长因子(VEGF)诱导CXCR4+BMSCs分化,采用流式细胞仪检测基因表型；用实时定量RT-PCR和FACS分别检测内皮细胞(EC)表记物PEC AM-1和vWF的mRNA和蛋白表达水平；CXCR4+BMSCs功能性作用通过检测是否摄取乙酰化低密度脂蛋白(ac-LDL)和血管形成能力来评价；最后通过小鼠肢体缺血模型移植CXCR4+BMSCs,观察其对梗死区新生血管形成和修复的影响。结果：在VEGF诱导下,CXCR4+BMSCs可表达EC表面标志物Flk-1,ICAM-1, VCAM-1。与对照组相比,VEGF显著提高CXCR4+BMSCs中PECAM-1和vWF的mRNA和蛋白表达水平。在VEGF诱导下,CXCR4+BMSCs能摄取Dil标记的ac-LDL。在Matrigel胶上CXCR4+BMSCs能形成血管样结构,VEGF显著促进了管样结构的形成。电镜下CXCR4+BMSCs具有EC样连接结构和EC特征性Weibel-Palade样的超微结构。最后,在小鼠肢体缺血模型移植CXCR4+BMSCs中,CXCR4+BMSCs参与新生血管的形成,促进了梗死区域修复。结论：结果证实在VEGF诱导下CXCR4+BMSCs可分化成EC样细胞,参与损伤后新生血管形成,对损伤后修复具有重要的治疗意义。更多还原

【Abstract】 Background:Bone mesenchymal stem cells (BMSCs) are attractive candidates for cell based therapies to cardiovascular disease such as infarction and atherosclerosis; however, the mechanisms responsible for stem cell chemotaxis and homing remain unknown. Chemokine stromal cell-derived factor 1 (SDF-1a) is involved in the process of atherogenesis. This study was aimed at investigating whether the SDF-1a of human umbilical vein endothelial cells (HUVECs) play a role in migration of BM-derived CXCR4+(receptor for SDF-la) stem cells.Methods:HUVECs were isolated and cultured from human umbilical cords and were treated with ox-LDL. The mRNA and protein expression of SDF-la was detected in HUVECs. CXCR4+BMSCs from bone marrow were isolated and were tested by migration and adhesion assays.Results:It was found that ox-LDL induced HUVECs to increase the mRNA and protein expression of SDF-1a. In addition, ox-LDL induced HUVECs to increase the expression of hypoxia-inducible factor-1 (HIF-1a). Ox-LDL enhanced the migratory and adhesion response of CXCR4+BMSCs. When the neutralizing SDF-la antibody abrogated the secreted SDF-la, the migration and adhesion response of CXCR4+BMSCs markedly was inhibited.Conclusions:Our data indicated that the endothelial cells (ECs) stimulated by ox-LDL could increase the BMSCs migratory response via SDF-la/CXCR4 signaling axis. These findings provide a new paradigm for biological effects of ox-LDL and have implications for novel stem cell therapeutic strategies for atherosclerosis. Background:Recent findings indicate that bone marrow mesenchymal stem cells (BMSCs) participate in the process of neovascularization in response to repair-to-injury and are involved in post-infarction myocardial repair. CXCR4+ stem/progenitor cells mobilized to the infarct area and improved the myocardial repair. It is unclear what special characteristics the vascular progenitors of bone marrow origin have. In present study, we aimed to determine whether CXCR4+BMSCs contribute to the angiogenic capacity in vitro and in vivo.Methods:CXCR4+BMSCs were separated by using paramagnetic microbeads and cultured. RT-PCR and FACS analysis confirmed the gene expression phenotype. The uptake of acetylated low density lipoprotein (ac-LDL) and the capillary tube formation in Materigel were evaluated. The effect of CXCR4+BMSCs transplantation on neovascularization was investigated in a murine model hindlimb ischemia.Results:After induced by VEGF, CXCR4+BMSCs expressed the endothelial cells (ECs) phenotype. The expression of EC markers, PECAM-1 and von Willebrand factor (vWF) increased significantly at both the mRNA and protein levels. In addition, CXCR4+BMSCs enhanced the uptakes of Dil-ac-LDL and form capillary-like tubes in vitro. In vivo the local transfer of CXCR4+BMSCs increased neovascularization in ischemic hindlimb.Conclusion:These results demonstrate that CXCR4+BMSCs differentiate into ECs and contribute to neovascularization in the vascular lesion, which indicate the important therapeutic implications for repair-to-injury and a new cell source for cell-based vascular engineering in the future.更多还原