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

1.确定用于从骨髓细胞进行血管再生的有效和简单的细胞群分选：以临床应用为目标，在其他研究资料的基础上，G-CSF动员的外周血单核细胞被认为是“用于从骨髓细胞再生血管的细胞群”的替代物，并被用于实验“2“。在体外结合骨髓细胞、生长因子和人工细胞外基质（ECM）这三种不可避免的元素用于血管再生：本研究的主要主题。在含有I型和IV型胶原、纤连蛋白、玻连蛋白和纤维蛋白原作为ECM的培养基中观察到培养板中骨髓来源的CD 34阳性细胞的内皮定殖。术后第1年纤连蛋白的疗效是其他ECM的5倍。在第2年，为了通过三维培养进一步离体血管再生，将临床使用的人造血管之一PTFE（Polyurethane）片材在涂覆PAU（Polyaminourethane）以使其亲水并增加组织亲和力后用于细胞培养“支架”，使用骨髓来源的CD 34阳性细胞和G-CSF动员的外周血单个核细胞。6种ECM、I、IV型胶原、纤连蛋白、2种纤连蛋白肽和玻连蛋白在三维培养体系中对细胞粘附和增殖的促进作用，其中以纤连蛋白RGD肽的促进作用最强。3.分析“血管片”中内皮细胞和平滑肌细胞转分化的机制尚不清楚。

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.