兔骨髓间充质干细胞的体外分离培养及鉴定

目的：观察兔骨髓间充质干细胞（BMSCs）生长特性及潜在分化潜能,为组织工程中种子细胞选择提供实验基础。方法：应用全骨髓贴壁法分离培养兔BMSCs,相差显微镜下观察其生长特点,应用流式细胞术对第三代细胞进行表面抗原鉴定。经成脂和成骨诱导液体外诱导兔BMSCs向脂肪细胞、成骨细胞分化,对诱导2周后的细胞进行油红O染色、茜素红染色、Van-kossa银染染色及碱性磷酸酶染色。结果：经流式细胞术鉴定,全骨髓贴壁法可获得兔BMSCs,第三代兔BMSCs生物特征基本一致并能诱导分化为脂肪细胞及成骨细胞,成脂诱导后油红O染色细胞内出现红色脂滴,成骨诱导后茜素红染色、Vankossa银染均可观察到矿化结节。碱性磷酸酶活性染色对照组呈弱阳性,诱导组呈强阳性。结论：全骨髓贴壁法是分离培养兔BM-SCs简便可行的方法。BMSCs来源丰富并有成脂及成骨潜能,是组织工程的优良种子细胞。

ISOLATION,CULTURE AND IDENTIFICATION OF MESENCHYMAL STEM CELLS FROM THE BONE MARROW OF RABBIT IN VITRO

Objective：The rabbit bone marrow mesenchymal stem cells（MSCs） were observed the biological characteristics and the differentiation potential,which to provide experimental basis for selection in the seed cells for tissue engineering.Methods：The rabbit bone marrow-derived MSCs were isolated and cultured from rabbit bone marrow by the bone marrow different adherent method.Morphology of MSCs was examined by phase contrast microscopy,surface antigen from the third passage MSCs was detected by flow cytometry.MSCs were treated with adipose inductor and osteogenetic inductor to differentiated into adipocytes and osteoblast in vitro.And the differentiated cells were identified by oil red O staining,alizarin bordeaux staining,Vankossa silver staining and alkaline phosphatase staining.Results：Through flow cytometry analyzed,the bone marrow-derived MSCs were obtained by the different adherent method.The biological characteristics of 3 passage MSCs were consistent,which were induced into adipocytes and osteoblast.After adipose induction,oil red O staining showed that red lipid droplet existed in adipose cells.After osteogenetic induction,mineralized nodules were detected by alizarin Bordeaux staining and Vankossa silver staining,the controlled cells were weak positive to alkaline phosphates staining.The induced-cells were strong positive.Conclusion：The different adherent method is a simple and feasible way,which isolate and culture MSCs from rabbit bone marrow.The MSCs are superior seed cells of tissue engineering,which have rich sources and differentiation potential into adipocytes and osteoblast.