EphrinB2基因工程的骨髓间质干细胞对分化成血管内皮细胞的影响

EphrinB2 gene transfection promotes the differentiation of bone marrow mesenchymal stem cells into vascular endothelial cells

目的观察研究ephrinB2基因转染对体外诱导环境大鼠骨髓间质干细胞(BMSCs)分化成血管内皮细胞的促进作用。方法采用密度梯度离心-贴壁培养法从Wistar大鼠骨髓中得到BMSCs。采用lenti-virus载体装载人ephrinB2基因转染大鼠BMSCs。采用流式细胞计数测定CD105、CD73、CD44、八因子(VWF)和血管内皮生长因子受体(KDR)等标志物在ephrinB2-BMSCs中的表达,并探讨其向成骨细胞及脂肪细胞体外分化的多向分化潜能。以2%FBS和50ng/ml血管内皮生长因子(VEGF)的培养条件,体外诱导ephrinB2-BMSCs向血管内皮细胞分化,并测定相应标志物的表达。结果未经诱导分化的ephrinB2-BMSCs表达CD105、CD73和CD44,不表达血管内皮细胞特异性标志物VWF和KDR。转染后的ephrinB2-BMSCs依然具有分化成脂肪细胞和成骨细胞的多向分化潜能。经诱导,ephrinB2-BMSCs表达VWF与KDR,并且其生成血管内皮细胞的比率及在Matrix基质上形成毛细血管样结构的比率均显著高于未转染ephrinB2的BMSCs。结论ephrinB2基因工程的BMSCs具有极高的向血管内皮细胞分化的潜能。这种基因工程细胞为建立冠心病患者的临床治疗新方法提供了有意义的资源。

Objective To study the effects ofephrinB2 gene transfection on the differentiation of bone marrow mesenchymal stem cells （BMSCs） into vascular endothelial cells. Methods Wistar rat BMSCs were isolated by density gradient centrifugation and purified on the basis of their adhesion ability. The BMSCs were transfected with a lenti-virus vector encoding a constitutively active form of human ephrinB2 gene, and the cell markers including CD105, CD73, CD44, von Willebrand factor （VWF） and vascular growth factor receptor 2 （KDR） were detected using flow cytometry. The potential of ephrinB2-BMSCs for differentiation into osteoblasts and adipoblasts in vitro were tested, and the differentiation of the cells into endothelial-like cells was induced by culture in the presence of 2% fetal bovine serum and 50 ng/ml vascular endothelial growth factor. Results EphrinB2-BMSCs were positive for the markers CD105, CD73 and CD44, and negative for the typical endothelial markers like VWF and KDR, and retained high potentials for differentiation into osteoblasts and adipoblasts in vitroafter cultivation in respective media. After induced differentiation, ephrinB2-BMSCs expressed VWF and KDR and showed greater ability of differentiation into vascular endothelial cells and formation of capillary structures on matrix gel than the BMSCs without transfection. Conclusions EphrinB2 gene transfection efficiently promotes the differentiation of BMSCs into vascular endothelial cells. These genetically engineered cells provide valuable sources for new therapies of coronary heart disease.