大鼠骨髓间充质干细胞分离培养及多向分化研究的体外实验

Isolation and cultivation of rat bone marrow mesenchymal stem cells and in vitro experiment of multi-differentiation potential

目的分离培养较高纯度大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs),检测其多向分化潜能。方法采用密度梯度离心结合差速贴壁法分离培养大鼠BMSCs,观察细胞形态,检测表面标志物CD34、CD44、CD45、CD90表达情况,及其成骨、成脂、成神经诱导后茜素红染色、油红O染色和NSE、GFAP免疫荧光染色情况。结果细胞呈典型的成纤维细胞样形态,CD44和CD90呈阳性表达,CD34和CD45呈阴性表达,细胞纯度>98%。成骨、成脂、成神经诱导后,茜素红染色、油红O染色、NSE和GFAP免疫荧光均为阳性。结论密度梯度离心结合差速贴壁法可分离、培养出高纯度的大鼠BMSCs,该细胞具有成脂、成骨、成神经多向分化潜能,是脊髓损伤修复的一种较理想的种子细胞。

Objective To isolate and cultur high pure rat bone marrow mesenchymal stern cells （BMSCs） and investigate the osteogenic, adipogenic and neurogenic differentiation potential in vitro. Methods Rat BMSCs were isolated by density gradient centrifugation combined with differential adhesion, the morphological characteristics were observed by inverted micro scope, and the surface markers （ CD34, CD44, CD45, CD90） were examined by flow cytometry. Rat BMSCs were induced to differentiated toward to osteoblasts, adipocytes and neural cells, and the ＂,alizarin red staining, oil red O staining and NSE, GFAP immunofluorescence were determined respectively. Results Rat BMSCs possessed a typical fibroblast-like shape, the expression of CD44 and CD90 was positive, and the expression of CD34 and CIM5 was negative, the purity was 〉 98%. After osteogenic, adipogenic and neurogenic inducing, the rat BMSCs showed positive reactions for alizarin red staining, oil red O staining and NSE and GFAP immunofluorescence respectively. Conclusion High pure rat BMSCs were successfully isolated and cultured by density gradient centrifugation combined with differential adhesion, and possessed multi-differentiation poten-tial. It shows great promise for spinal cord injury repairing applications.