全骨髓贴壁法培养兔骨髓间充质干细胞体外定向成骨诱导分化及鉴定

In vitro osteogenic differentiation and identification of rabbit bone marrow mesenchymal stem cells isolated and cultured with whole bone marrow adherent culture method

背景:流式细胞仪分离法和免疫磁珠分离法对细胞活性影响较大,密度梯度离心法虽然能够获得纯度高的单核细胞,但由于多次离心可造成细胞的大量流失且对细胞活性有一定的影响使其应用值得商榷。目的:采用全骨髓贴壁法分离兔骨髓间充质干细胞进行成骨诱导分化及鉴定。方法:采用全骨髓贴壁法体外分离培养兔骨髓间充质干细胞,倒置显微镜下观察细胞形态学特征。在成骨诱导剂作用下,通过碱性磷酸酶染色试剂盒行碱性磷酸酶染色,Ⅰ型胶原免疫细胞化学染色,VonKossa法及茜素红进行矿化结节染色以及电镜下检测兔骨髓间充质干细胞成骨诱导后的形态结构。结果与结论:经诱导后细胞出现与成骨细胞相似的形态学特征,碱性磷酸酶染色阳性,Ⅰ型胶原免疫细胞化学染色,Von-Kossa法及茜素红矿化结节染色阳性。表明经成骨诱导剂诱导后全骨髓贴壁法体外分离纯化培养的兔骨髓间充质干细胞能向成骨细胞方向分化增殖。

BACKGROUND： The flow cytometry and immunomagnetic separation method have greater impact on ceil viability, and although the density gradient centrifugation method can be used to obtain the high purity monocytes, however, the multiple centrifugation may cause a huge loss of cells and has some impact on the cell activity, so the application is debatable. OBJECTIVE： To isolate and culture rabbit bone marrow mesenchymal stem cells with whole bone marrow adherent culture method for osteogenic differentiation and identification. METHODS： The rabbit bone marrow mesenchymal stem cells were isolated and cultured by whole bone marrow adherent culture method, then the morphological characteristics of rabbit bone marrow mesenchymal stem cells were observed under inverted microscope. Under the induction of osteogenicinducer, alkaline phosphatase staining, type I collage immunocytochemical staining, alizadn red staining and Von-Kossa staining were performed to observe the morphology of rabbit bone marrow mesenchymal stem cells after osteogenic induction by electron microscope. RESULTS AND CONCLUSION： After in v#ro induced differentiation, rabbit bone marrow mesenchymal stem cells exhibited the morphological and biological characteristics similar to typical osteoblasts, alkaline phosphatase staining, type I collagen immunocytochemical staining, and Von-Kossa staining and alizadn red mineralization nodules staining were positive. All results indicate that rabbit bone marrow mesenchymat stem cells in vitro isolated and cultured with whole bone marrow adherent culture method can be induced to differentiate into osteoblasts after osteogenic induction.