体外诱导骨髓基质干细胞向内皮细胞分化的实验研究

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：骨髓基质干细胞表达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。

In vitro induction of bone marrow stromal stem cells to differentiate into endothelial cells

Objective: To investigate the feasibility of bone marrow stromal stem cells(BMSCs) differentiating into en-dothelial cells (ECs) in vitro,providing new cell sources for cardiovascular tissue engineering. Methods:Sheep bone marrow was aspirated from iliac crest. BMSCs were isolated by centrifugation through a Ficoll step gradient .purified by plating culture and depletion of the non-adherent cells. Primary BMSCs were cultured in M199 medium supplemented with basic fibroblast growth factor (bFGF) .epidermal growth factor (EGF) and insulin-like growth factor (IGF),and were induced to differentiate into ECs with vascular endothelial growth factor (VEGF).The phenotypes of differentiated cells were identified by flow cy-tometry analysis of CD34.CD31 antigen expression and immunocytochemical staining of CD34.CD31 and Factor VIII-related antigen. Their functions were evaluated by UEA binding test and NO release analysis. Results:BMSCs expressed a-SMA and were negative of CD34 and Factor Vl-related antigen. After 2 weeks of committed induction BMSCs differentiated into spindle-shaped ECs. The differentiated cells expressed CD34.CD31 and Factor Vl-related antigen; they also had the capability of binding UEA and releasing NO. Conclusion :BMSC can be induced to differentiate into ECs in vitro; the differentiated cells have the same characteristics and functions as mature ECs.