兔骨髓内皮祖细胞在组织工程血管构建中的实验研究

摘要

To establish the method for isolating, cultivating and identificab'ng of rabbit endothelial progenitor cell in vitro and investigate the function of EPCs in tissue engineering blood vessel construction. EPCs were obtained by mononuclear cells which were separated by density gradient centrifugan'on and were seeded on the inner surface of tissue engineered graft with human fibronectin(FN) as seed cells, then in vitro induction culture by vascular endothelial growth factoKVEGF) and basic fibroblast growth factor (bFGF). In control group no VEGF, bFGF and FN were added. Tissue engineered graft were identified and analyzed. Cultured bone marrow mononuclear cells were showed on 'slabstone shaped' and identified as endothelial progenitor cell by immunofluorescence and phagocytosis function tests. The cells planting density of tissue engineered graft was higher than that of control group at the 10* day seeding and culture in vitro. Endothelial cells were covered on the surface of vascular lumen completely by SUM examination and were survival uniformly in the tissue engineering blood vessel by H - E staining. EPCs were differentiated to mature vascular endothelial cells and were expressed VEGFR - 2, vWF, CD34 by imiminohistochenristry. Rabbit bone marrow mononuclear cells may differentiate to EPCs. VEGF, bFGF and FN may be beneficial to EPCs differentiation and proliferation in tissueengineering blood vessel, which create further conditions for small diameter tissue engineering blood vessel.%建立体外分离、培养及鉴定兔骨髓血内皮祖细胞(endothelial progenitor cell,EPCs)的方法,并探讨其在血管组织工程构建过程中的功能.采用密度梯度离心法分离单个核细胞,经培养鉴定为EPCs后作为种子细胞接种于人纤维连接蛋白包被(FN)的组织工程血管支架上,加入血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)进行体外诱导培养,同时设置未包被纤维连接蛋白及未添加血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)的培养方法作为对照组,体外培养10 d后,对构建的组织工程血管进行鉴定分析.分离培养的骨髓单个核细胞呈典型的“铺路石样”外观.经免疫荧光检测、细胞吞噬功能鉴定为内皮祖细胞；种植细胞10 d后结果显示:加入纤维连接蛋白和血管内皮生长因子的血管支架可见细胞种植密度明显高于对照组,扫描电子显微镜观察到,血管内腔面较为完整的覆盖内皮细胞.HE染色显示:内皮细胞在血管支架上成活并较为均匀；免疫组化结果显示分化为成熟血管内皮细胞并表达VEGFR-2、vWF、CD34.兔骨髓单个核细胞体外培养可以诱导分化为内皮祖细胞,血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)和纤维连接蛋白(FN)的组合更有利于内皮祖细胞在血管支架上增殖和分化,为人工血管制备创造了条件.