大鼠骨髓间充质干细胞的体外分离培养与鉴定

Isolation,Culture and Identification of Rat Mesenchymal Stem Cells In Vitro

目的建立大鼠骨髓间充质干细胞(Mesenchymal stem cells,MSCs)体外分离培养及鉴定的方法 ,为MSCs的系列研究奠定基础。方法采用全骨髓直接贴壁筛选法分离培养MSCs并传代,倒置相差显微镜下观察细胞形态,以MTT法检测细胞增殖水平并绘制生长曲线。取第3代MSCs,流式细胞术检测细胞周期和细胞表型,应用成骨细胞诱导液和脂肪样细胞诱导液诱导MSCs定向分化,鉴定其分化能力。结果全骨髓细胞培养5d,镜下可见贴壁细胞增殖明显,细胞形态较均一,大部分呈梭形,7d左右可传代,经2～3次传代后细胞呈单一梭形的成纤维样细胞,即MSCs;细胞生长曲线呈S形;经流式细胞仪检测,MSCs细胞76.01%处于G0/G1期,7.13%处于G2/M期,16.86%处于S期;MSCs表面不表达CD34;在特定诱导液作用下,MSCs可分别向成骨样细胞及脂肪样细胞分化。结论已成功建立了分离培养及鉴定MSCs的方法 ,可用来评价体外培养的MSCs。

Objective To develop the methods for isolation,culture and identification of rat mesenchymal stem cells（MSCs） in vitro and lay a foundation of a serial study on MSCs.Methods MSCs were isolated and cultured by direct wall adhesion culture of the whole bone marrow,then subcultured and observed for morphology under invert microscope.The proliferation of MSCs was determined by MTT method,based on which a growth curved was plotted.The MSCs of passage 3 were determined for cell cycle and phynotype by flow cytometry.The directional differentiation of MSCs to osteoblasts and fat-like cells were induced,and the differentiation ability was identified.Results After the whole bone marrow cells were cultured for 5 d,obvious proliferation of adherent cells were observed under microscope,and the cells were even in shape,most of which were spindle-shaped.The cells could be subcultured about 7 d after culture,and formed spindle-shaped fibroblast-like cells,i.e.MSCs after subculture for 2 ~ 3 passages.The growth curve of cells was in S shape.Flow cytometry showed that 76.01%,7.13% and 16.86% of MSCs were in G0 /G1,G2 /M and S phases respectively.No CD34 was expressed on the surface of MSCs.However,by using specified inducers,MSCs were differentiated to fibroblast-like and fat-like cells respectively.Conclusion The methods for isolation,culture and identification of rat MSCs in vitro was successfully developed,which might be used for evaluation of MSCs cultured in vitro.