兔骨髓间充质干细胞培养与多向分化潜能鉴定的实验研究

目的原代培养兔骨髓间充质干细胞(mesenchymal stem cells,MSCs),观察体外培养中MSCs的生物学特性及多向分化潜能。方法通过密度梯度离心联合贴壁培养法,体外分离、纯化、扩增兔骨髓间充质干细胞,观察形态学特点,测绘传代MSCs生长曲线,检测细胞周期及表面标记,体外诱导MSCs向成骨细胞、成脂肪细胞分化并鉴定。结果原代及传代MSCs为长梭形成纤维细胞样细胞;生长曲线显示传代细胞具有类似的生长规律;流式细胞仪分析MSCs CD44表达阳性,CD45表达阴性;细胞周期分析显示87%以上细胞处于G0/G1期;经成骨细胞诱导,细胞碱性磷酸酶染色阳性;经成脂肪细胞诱导,细胞内出现红染脂滴。结论通过密度梯度离心联合贴壁培养法可大量扩增、纯化MSCs,所获细胞具有高度自我更新和多向分化潜能。

Experimental study of primary culture and multi-directional differentiation of rabbit bone marrow derived mesenchymai stem cells in vitro

Objective To culture bone marrow derived MSCs (mesenchymal stem cells) in vitro,and then observe its biological characteristics and potentiality of multi-directional differentiation.Methods To isolate,purify and amplify the MSCs from rabbit bone marrow by density gradient centrifugation and adherent culture in vitro,observe the characteristics of MSCs morphology by inverted phase contrast microscope and transmission electron microscope ;survey and map MSCs growth curve ;detect cell membrane's marks and cell cycle by flow cytometer;MSCs were induced into osteogenetic cells in the osteogenesis supplement medium or adipose cells in adipogenesis supplement medium,and then they were verified.Results The primary cells and the passage cells were mostly fusiform in shape,to be similar to fibroblast cell.the growth curve showed the growth regularity of the passage cells was similar; the flow cytometer analysis showed that the membrane mark CD44 was positive,CD45 was negative; the cell cycle analysis showed that more than 87% cells were in G0/G1 phase; after MSCs were induced,cell alkaline phosphatase stain showed that alkaline phosphatase was positive in osteogenetic cell,oil red O stain showed that red lipid droplet existed in adipose cell.Conclusion MSCs can be isolated and cultured by the method of density gradient centrifugation and adherent culture in vitro,they have the better potentiality of proliferation and multi-directional differentiation.