用骨髓基质干细胞分化的内皮细胞体外构建组织工程心脏瓣膜

In vitro construction of tissue-engineered heart valves with endothelial cells differentiated from bone marrow mesenchymal stem cells

目的:探讨用骨髓基质干细胞(BMSCs)诱导分化产生的内皮细胞和去细胞异种天然瓣膜支架体外构建组织工程心脏瓣膜(TEHV)的可行性。方法:以含EGF、bFGF、IGF和肝素的M199培养液培养绵羊原代BMSCs,并以VEGF诱导BMSCs向内皮细胞分化。采用去污剂和酶消化法制作去细胞猪主动脉瓣支架,通过静态种植方法构建TEHV。经H-E染色、免疫组化、扫描电镜及透射电镜检查观察TEHV的组织学和超微结构。结果:诱导分化产生的内皮细胞在去细胞瓣叶支架及整体瓣膜支架上呈单层生长,形成完整的内皮细胞单层。瓣叶表面细胞呈梭形,CD34及Ⅷ因子相关抗原免疫组化染色阳性。结论:BMSCs诱导分化产生的内皮细胞具有与成熟内皮细胞相同的生物学特性。采用BMSCs诱导分化内皮细胞构建TEHV更为简便可行。

Objective：To investigate the feasibility of constructing a tissue-engineered heart valve （TEHV） with endothelial cells differentiated from bone marrow mesenchymal stem cells （BMSCs） and acellular porcine aortic valve matrices. Methods： Primary ovine BMSCs were cultured in M199 medium supplemented with basic fibroblast growth factor （bFGF）, epidermal growth factor （EGF）, insulin like growth factor （IGF） and heparin, and were induced to differentiate into endothelial cells with vascular endothelial growth factor （VEGF）. Decellularized valve matrices were developed from porcine aortic valves treated with detergent and enzymatic procedure. TEHV was constructed by seeding differentiated endothelial cells onto acellular matrices. Morphological characteristics of TEHV were evaluated by histological and electron microscopic observations. Results： H-E staining, scanning electron microscopy （SEM） and transmission electron microscopy （TEM） demonstrated that the surface of tissue-engineered valve leaflets and the inner surface of tissue-engineered valve conduits were confluent, covered with a monolayer of spindle-shaped cells positive of CD34 and factor VE related antigen. Conclusion： Endothelial cells differentiated from BMSCs have the same biological characteristics as mature endothelial cells. Development of TEHV with differentiated endothelial cells （induced by BMSCs） is feasible and simple.