兔骨髓间充质干细胞的分离、培养及鉴定

Isolation、Culture and Identification of Rabbit Bone Marrow derived Mesenchymal Stem Cells

目的探讨兔骨髓间充质干细胞分离、培养和鉴定的方法。方法全骨髓贴壁法提取兔骨髓中的间充质干细胞,流式细胞术检测细胞表面分子CD14、CD29、CD34、CD44、CD45和CD90等标志的表达以及不同代次兔BM-SCsDNA含量。通过定向分化检测兔BMSCs的多向分化潜能。结果兔骨髓间充质干细胞呈形态较为均一的小梭形、克隆样生长。流式细胞术检测结果显示,传代至第12代和20代的兔骨髓间充质干细胞检测对数生长期细胞的DNA含量结果显示细胞为正常二倍体细胞,无明显变异。细胞表面表达CD29(61.71%)、CD44(60.2%),不表达CD14(0.4%)、CD34(0.34%)、CD45(0.64%)和CD90(0.04%)。兔BMSCs的定向分化结果显示其可向成骨细胞、脂肪细胞及成软骨细胞分化。结论成功获得具有多向分化潜能的兔骨髓间充质干细胞,其具有特定的细胞表面抗原,具有容易获取、增殖能力强等特点,是优良的组织工程种子细胞。

Objective To evaluate the methods of isolation,culture and identification of rabbit bone marrow derived mesenchymal stem cells（BMSCs）.Methods The rabbit BMSCs were separated by the whole bone marrow adherence method,which were analyzed the cell surface molecules CD14,CD29,CD34,CD44,CD45and CD90expression and cell DNA contents in different generations by Flow cytometry.We evaluated the multi-directional differentiation ability of BMSCs through the method of directed differentiation.Results The rabbit BMSCs cells were homogeneous spindleshaped in a phase contrast microscope.Flow cytometry results indicated that cells were all diploid of passage 12and passage 20,there were no obvious variation.Flow cytometry results also showed the Cell surface antigen CD29（61.71%）、CD44（60.2%）expressed on the BMSCs,and there were no expression of CD14（0.4%）、CD34（0.34%）、 CD45（0.64%）and CD90（0.04%）.The directed differentiation results showed the BMSCs could differentiate into Osteoblasts,fat cells and chondrocytes.Conclusion We successfully cultured the rabbit BMSCs which had multi-directional differentiation ability and specific cell surface antigens.The BMSCs could easily got and proliferated,it’s a excellent tissue engineering seed cell.