糖尿病大鼠骨髓内皮祖细胞的培养与鉴定

Culture and Identification of Endothelial Progenitor Cells from Bone Marrow of Diabetic Rat

目的探讨糖尿病大鼠骨髓来源的内皮祖细胞体外诱导、培养和扩增的方法。方法用密度梯度离心法采集糖尿病大鼠骨髓单个核细胞,在含有血管内皮生长因子和碱性成纤维细胞生长因子的M199培养基中进行体外培养。应用免疫组织化学和免疫荧光法鉴定内皮祖细胞的CD34和CD133,并通过检测其对FITC标记的UEA-1的吸附和Dil-acLDL的吞噬来进行细胞功能的鉴定。结果在培养2天后部分细胞开始贴壁、变大,并逐渐伸展呈梭形。第7天时贴壁细胞增生明显,"集落"样生长。培养第7天细胞CD34和CD133均呈阳性,激光共聚焦显微镜观察显示EPC可以同时吞噬ac-LDL并结合UEA-1。结论糖尿病大鼠骨髓可以分离培养出内皮祖细胞,并能体外扩增,为糖尿病内皮祖细胞的移植研究奠定了基础。

ObjectiveTo explore the induction, training and expansion in vitro of endothelial progenitor cells from bone marrow of diabetic rat. MethodsMononuclear cells were harvested from bone marrow of diabetic rat by density gradient centrifugation and were induced in M199 medium containing VEGF and bFGF. The specific protein expression of endothelial progenitor cells for CD34 and CD133 were observed by immunohistochemistry and immunofluorescence.The biological functions of endothelial progenitor cells were examined by the adsorption of Ulex Europaeus Agglutinin（UEA） labeled by fluorescein isothiocyanate（FITC） and DiI labeled by acetylated low density lipoprotein internalization.ResultsCultured cells showed the characteristic of adherence and were gradually extended spindle in the second days after the culture.In the 7th day, the adherent cells proliferated faster and exhibited the clone-like morphology and were positive for CD34 and CD133.The cells could take up DiI-acl DL and bind to FITC-UEA-1,which showed double positive fluorescence under LSCM. ConclusionThe mononuclear cells from bone marrow of diabetic can be differentiated into EPCs under certain condition,which provides the basic for clinical application of endothelial progenitor cells transplantations.