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

1.确定有效、简便的骨髓血管再生细胞群分选方法：针对临床应用，结合近期其他研究资料，认为G-CSF动员外周血单个核细胞可替代“骨髓血管再生细胞群”，用于实验“2”2。体外联合骨髓细胞、生长因子和人工细胞外基质（ECM）三种血管再生不可缺少的元素，确定短期“血管片”的最佳制备条件：本研究的主题。在以I型和IV型胶原蛋白、纤维连接蛋白、玻璃体连接蛋白和纤维蛋白原为ECM的培养基中，观察到骨髓来源的CD34阳性细胞在培养板上的内皮定植。治疗1年后，纤维连接蛋白的疗效是其他ECM的5倍。第二年，为了进一步进行体外血管三维培养再生，采用临床常用的血管假体之一聚四氟乙烯（PTFE）片作为细胞培养“支架”，用PAU（聚氨基甲酸乙酯）包被，使其具有亲水性，增加组织亲和力，骨髓来源的CD34阳性细胞和G-CSF动员的外周血单个核细胞。采用6种ECM、I型和IV型胶原蛋白、纤维连接蛋白、2型纤维连接蛋白肽和玻璃体连接蛋白，观察其在三维培养体系中对细胞粘附和增殖的加速作用，其中以纤维连接蛋白RGD肽最有效。分析“血管片”中内皮细胞与平滑肌细胞的转分化机制尚不清楚。

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.