体外诱导小鼠骨髓单个核细胞向内皮分化的实验研究

Differentiation of Mouse BM-MNCs into Endothelial Cells in vitro

目的:为组织工程研究做准备,分离培养小鼠骨髓单个核细胞并诱导向血管内皮分化。方法:成年Balb/c小鼠,应用淋巴细胞分离液(密度1.077g/m l),将长骨骨髓经F icoll非连续密度梯度离心,收集中层的单个核细胞,加入诱导培养基并置于纤维连接素包被的培养板上进行诱导分化,观察细胞生长状况,以透射电镜及Ⅷ因子、CD31、Lectin免疫组化以及流式细胞仪方法对培养细胞进行鉴定。结果:细胞圆形、纺锤形单层融合贴壁生长;Ⅷ因子、CD31、Lectin免疫组化染色阳性;透射电镜显示细胞具有内皮特征性的W-P小体。结论:采用F icoll密度梯度离心可获得较高纯度的骨髓单个核细胞,经体外培养并诱导分化,具有血管内皮细胞的特征。

Objective：To culture and induce endothelial cells from mouse bone marrow in vitro and prepare for the vascular tissue engineering research. Methods： Bone marrow cell suspension of adult mouse was prepared by discontinuous gradient centrifugation on Ficoll（density 1. 074g/ml） , mononeuclear cells in the middle layer was collected, put into selective culture medium and cultured in the fibernectin coated plate. The cells were observed under inverted microscope every day. The cells was identified by transmission electron microscope, flow cytometer and immunohistochemical staining with factor Ⅷ, CD31, Lectin antigen. Results：The cells had round or spindle shape. The cells were factor Ⅷ, Lectin and VEGFR positive. Under transmission electron microscope, the cells displayed Weibel - Palade bodies which were endothelial character. Conclusion： This study cultures and induces mouse bone marrow EPCs successfully in vitro. Discontinuous density gradient centrifugation is effective approach to isolating mouse bone marrow stromal cells. The cells induced in vitro have the same characters as endothelial cells.