大鼠骨髓间充质干细胞分离鉴定方法及生物学特性

Isolation, identification and biological characteristics of rat bone marrow mesenchymal stem cells

背景:如何能简便高效地获得均质性的骨髓间充质干细胞群是软骨组织工程研究的重点。目的:分离培养大鼠骨髓间充质干细胞,观察其生物学特性。方法:抽取30只SD大鼠股骨以及胫骨骨髓,采用全骨髓贴壁培养法体外分离培养骨髓间充质干细胞,流式细胞仪测定细胞表面标记物的表达,细胞计数法观察细胞生长增殖能力,MTT法检测细胞存活率。结果与结论:(1)刚分离培养的细胞呈圆形、椭圆形,部分细胞呈三角形,接种12-15 d后,细胞开始融合,传代后细胞增殖速度加快,传代2 h后细胞均匀分布在瓶底;(2)随着传代次数的增加,细胞增殖能力出现下降趋势,第1-5代细胞存活率均超过95%,显著高于第6代和第7代(P<0.05);(3)骨髓间充质干细胞表面CD34呈阴性表达,CD44呈阳性表达;(4)结果表明,采用全骨髓贴壁法可有效分离大鼠骨髓间充质干细胞,获得的细胞生长稳定,增殖能力强,可选用前5代细胞作为软骨组织工程的种子细胞。

BACKGROUND： How to effectively harvest bone marrow mesenchymal stem cells with homogeneity is crucial for cartilage tissue engineering research.OBJECTIVE：To isolate and culture rat bone marrow mesenchymal stem cells and to observe biological characteristics of the cells.METHODS：Bone marrow mesenchymal stem cells were isolated from the femur and tibia of 30 Sprague-Dawley rats using the whole bone marrow adhesion method.Cell surface markers were identified using flow cytometry,cell proliferation ability was observed through cell counting,and cell survival rate was determined by MTT assay.RESULTS AND CONCLUSION：The harvested cells were mostly round and oval,which partially showed a triangular shape.Cell fusion appeared after 12-15 days of inoculation.The proliferation of passaged cells was accelerated.Two hours after passage,the cells evenly covered the bottom of the culture dish.With the increasing of cell generations,the cell proliferation ability was gradually decreased.The survival rates of cells at passages 1-5 were over 95%,significantly higher than those at passages 6 and 7（P〈0.05）.The harvested cells were negative for CD34,but positive for CD44.To conclude,the whole bone marrow adhesion method is effective to isolate rat bone marrow mesenchymal stem cells with stable growth and strong proliferation ability.Passage 5 cells can be selected as seed cells for cartilage tissue engineering.