大鼠骨髓间充质干细胞的分离、培养及表型鉴定

目的 建立大鼠骨髓间充质干细胞分离及培养的方法,探讨体外培养骨髓间充质干细胞的生物学特性.方法 采用密度梯度离心法结合贴壁筛选法分离纯化大鼠骨髓间充质干细胞,传代扩增,测定生长曲线,镜下连续观察细胞的形态变化.流式细胞仪鉴定其表面抗原 CD29、CD34、CD44和CD45的表达情况.结果 原代骨髓间充质干细胞呈集落状生长,细胞呈梭形、纺锤形,呈放射状生长,传代后呈均一的成纤维细胞样.骨髓间充质干细胞生长性状相对稳定,1、3、5代细胞生长曲线基本一致.流式细胞仪鉴定表明,骨髓间充质干细胞CD44、CD29表达呈阳性,CD45、CD38表达呈阴性.结论 采用密度梯度离心法结合贴壁培养法能获得纯度较高的骨髓间充质干细胞,并且在体外培养条件下可大量增生,形成形态均一的细胞集落,可以作为组织工程中种子细胞的来源.

Isolation, culture and phenotype identification of rat marrow mesenchymal stem cells

Objective To establish a method of isolating and cultivating rat bone marrow mesenchymal stem cells (MSCs) in vitro and the biological characteristics thereof.Methods MSCs were isolated and purified from the bone marrow of rat by density gradient centrifugation and by adhering to the culture plastic.After successive subculture and amplification,the growth curve was drawn.The morphology of MSCs was observed in primary and passage culture under inverted microscope.The cells surface antigens of CD29,CD38,CD44 and CD45 were detected through flow cytometer.Results Cultured primary MSCs were spin dle-shaped and had a typical fibroblast-like morphology,proliferated in colonies.After a series of passage,the attached cells became morphological homogeneous and proliferated quickly.The growth curves of passage 1,3 and 5 were quite similar.The surface marker identification showed that CD44 and CD29 were positive,but CD45 andCD38 were negative.Conclusion The MSCs can be successfully isolated,cultured and purified from rat bone marrow through density gradient centrifugation,adherent property and subculture.MSCs can greatly proliferate after a short period of culture in vitro.The MSCs can be used as a potential cell source for tissue engineering.