Bone Marrow Cell Culture for Producing Tissue Engineered Regenerative Blood Vessels as a Goal of Clinical Application

1.骨髓细胞血管再生有效、简便分选细胞群的确定：针对临床应用，根据近期的研究资料，认为G-CSF动员的外周血单核细胞可替代“骨髓细胞血管再生细胞群”，用于实验2。体外快速制备“血管片”的最佳条件的确定结合了骨髓细胞、生长因子和人工细胞外基质三种不可缺少的成分。以I型和IV型胶原、纤维连接蛋白、玻璃体连接蛋白和纤维蛋白原为细胞外基质，观察骨髓CD34阳性细胞在培养板内的内皮定植情况。术后1年，纤维连接蛋白的疗效是其他ECM的5倍。第2年，为了通过三维培养进一步促进血管的体外再生，临床使用的血管假体之一聚四氟乙烯(PTFE)片被涂覆聚氨基尿烷(PAU)以形成亲水性和增加组织亲和力的支架用于细胞培养，使用骨髓来源的CD34阳性细胞和G-CSF动员的外周血单核细胞。在三维培养体系中，6种ECM、I型和IV型胶原、纤维连接蛋白、2纤维连接蛋白多肽和玻璃体连接蛋白对细胞黏附和增殖的促进作用进行了观察，其中以纤维连接蛋白RGD多肽效果最好。3.分析血管内皮细胞和血管内皮细胞间的跨分化机制尚不清楚。

1.Determination of effective and simple cell-groups sorting for vessel regeneration from bone marrow cells : Aiming at clinical applications, based on recent other research data, G-CSF mobilized peripheral-blood mononuclear cells are thought to be substituted for "cell-groups for vessel regeneration from bone marrow cells" and used for experiment "2"2.Determination of optimal conditions for making "vascular sheets" shortly In vitrocombining three inevitable elements, bone marrow cells, growth factors and artificial extracellular matrix (ECM) for vessel regeneration : Main theme of this study. Endothelial colonization of bone marrow derived CD34 positive cells in culture plates was observed in the media with type I & IV Collagen, Fibronectin, Vitronectin and Fibrinogen as ECM. It was obtained at 1st year that efficacy of Fibronectin was 5 times of those of other ECM. At 2nd year, aiming at further ex vivo vessel regeneration with 3 dimensional culture, one of clinically used vascular prosthesis, PTFE (Polytetrafluoroethylene) sheets, were used for cell culture "scaffold" after coating with PAU (Polyaminourethane) to make hydrophilic and to increase tissue affinity, Bone marrow derived CD34 positive cells and G-CSF mobilized peripheral-blood mononuclear cells were used. Six ECM, type I & IV Collagen, Fibronectin, 2 Fibronectin peptide and Vitronectin were used for observation of acceleration of cell adhesion and proliferation in 3 dimensional culture system Among them, Fibronectin RGD peptide was most efficient.3.To analyze the mechanism of transdifferenciation between endothelial cells and smooth muscle cells in "vascular sheets" It remains to be unclear.