大鼠骨髓基质干细胞体外诱导向内皮分化的实验研究

Experimental studies on induction of rat bone marrow stromal cells differentiating into endothelial cells

目的 分离培养大鼠骨髓基质干细胞(BMSCs),并诱导其向内皮分化,为组织工程研究作准备。方法 成年SD大鼠,应用Ficoll淋巴细胞分离液(密度为1.077 g/ml)将长骨骨髓经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,通过透射电镜观察、流式细胞仪检测阳性细胞百分率及Ⅷ因子、CD31、Lectin免疫组织化学检测,对培养细胞进行鉴定。结果 细胞呈圆形、纺锤形单层融合贴壁生长;Ⅷ因子、CD31、Lectin免疫组织化学染色阳性;透射电镜示细胞具有内皮特征性的Weibel Palade小体。结论 Ficoll密度梯度离心可获得较高纯度的BMSCs,经体外培养并诱导分化,具有血管内皮特征。

Objective To culture and induce endothelial cells from rat bone marrow in vitro, preparing for vascular tissue engineering research.Methods Bone marrow cell suspension of adult rat was prepared by discontinuous gradient centrifugation on Ficoll(density 1.077 g/ml), mononuclear cells in the middle layer was collected, and then put into selective culture medium and cultured in the fibernectin coated plate. The cells were identified by transmission electron microscope, flow cytometry and immunohistochemical staining with factor Ⅷ,CD31,Lectin antigen and were observed under inverted microscope every day. Results The cells were round or spindle shape, with factor Ⅷ-, Lectin- and VEGFR-positive. Under transmission electron microscope, the cells displayed Weibel-Palade bodies which was the characteristic of endothelium.Conclusion Discontinuous density gradient centrifugation is an effective approach to obtain highly pure rat bone marrow stromal cells. The cells induced in vitro have the same characteristics as the endothelial cells.