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目的:探讨成体骨髓基质干细胞体外定向诱导分化为内皮细胞的可行性,为心血管组织工程提供新的种子细胞来源。方法:骨髓穿刺抽取绵羊骨髓液,密度梯度离心法获取骨髓单个核细胞,通过贴壁培养纯化骨髓基质干细胞。以含EGF、bFGF、IGF和肝素的M199培养液培养原代细胞,并以VEGF诱导骨髓基质干细胞向内皮细胞分化。通过CD34、CD31流式细胞仪分析,免疫组化CD34、CD31、Ⅷ因子相关抗原染色鉴定内皮细胞,UEA结合实验及NO含量测定评价内皮细胞功能。结果:骨髓基质于细胞表达α-SMA,不表达CD34和CD31。体外定向诱导2周后分化为内皮细胞,呈梭形,表达CD34和CD31及Ⅷ因子相关抗原,UEA结合试验阳性,并具有分泌NO的功能。结论:体外培养条件下骨髓基质干细胞能够分化为内皮细胞,并具有成熟内皮细胞的生物学特性和功能。
OBJECTIVE: To investigate the feasibility of adult bone marrow stromal stem cells differentiating into endothelial cells in vitro and to provide a new source of seed cells for cardiovascular tissue engineering. METHODS: Bone marrow was collected by bone marrow aspiration and bone marrow mononuclear cells were obtained by density gradient centrifugation. Bone marrow stromal cells were purified by adherent culture. Primary cells were cultured in M199 medium containing EGF, bFGF, IGF and heparin, and VEGF was used to induce the differentiation of bone marrow stromal cells into endothelial cells. The function of endothelial cells was assessed by CD34, CD31 flow cytometry analysis, immunohistochemical staining of CD34, CD31, Ⅷ factor-related antigen staining endothelial cells, UEA binding assay and NO content determination. RESULTS: Bone marrow stromal cells express α-SMA in the cells but not CD34 and CD31. After 2 weeks of induced in vitro, the cells differentiated into endothelial cells, spindle-shaped, expressed CD34 and CD31 and factor Ⅷ related antigen, UEA binding test was positive, and had the function of secreting NO. CONCLUSION: BMSCs can differentiate into endothelial cells in vitro and have the biological characteristics and functions of mature endothelial cells.