兔骨髓间充质干细胞的分离培养及成骨分化的实验研究  

Experimental study on isolated culture and osteogenic differentiation of rabbit mesenchymal stem cells

作　　者：罗世君[1,2] 吴倩倩[1,2] 孙勇[2] 赵峰[2] 陈红亮[2] 

机构地区：[1]西南医科大学口腔医学院,四川泸州646000 [2]成都军区机关医院口腔科

出　　处：《西南国防医药》2016年第6期590-593,共4页Medical Journal of National Defending Forces in Southwest China

基　　金：全军“十二五”科研面上课题(CWS11J024)

摘　　要：目的观察兔骨髓间充质干细胞(BMSCs)的生物学特性及成骨分化潜能,为骨组织工程(BTE)临床应用提供实验基础。方法抽取兔骨髓(BM),采用密度梯度离心法结合贴壁筛选法分离培养兔BMSCs,通过流式细胞术(FCM)鉴定第3代(P3)BMSCs表面抗原CD44、CD45。随机将BMSCs分为实验组及对照组,实验组行成骨诱导培养,对照组行常规培养。实验组诱导3 w后行碱性磷酸酶(ALP)染色及茜素红染色,对照组常规培养后染色。结果密度梯度离心法结合贴壁筛选法可获得BMSCs,流式细胞术显示BMSCs高表达CD44(96.8%),低表达CD45(0.8%)。成骨诱导后细胞形态改变,茜素红染色可观察到矿化结节;ALP染色见细胞出现深蓝色钙化结节影。结论密度梯度离心法结合贴壁筛选法可较好的分离培养兔BMSCs。BMSCs经成骨诱导后向成骨细胞分化,可作为BTE理想的种子细胞。Objective To observe the biological features and osteogenic differentiation potential of rabbit-derived BMSCs and provide experimental basis for clinical application of bone tissue engineering（BTE）.Methods Rabbit BM was extracted; and BMSCs were isolated and cultured by density gradient centrifugation combined with attached screening method.Surface antigens CD44 and CD45 in P3 BMSCs were accredited by means of FCM.BMSCs were randomly divided into an experimental group and a control group.Osteogenesis induction culture was completed on the experimental group and conventional culture on the control group.ALP staining and alizarin red staining were completed for the experimental group three weeks after the induction culture and for the control group after conventional culture.Results BMSCs were obtained by density gradient centrifugation combined with attached screening method.FCM showed high expression of CD44（96.8%） and low expression of CD45（0.8%） in BMSCs.The cell morphology changed after osteogenic induction; nodules were observed by alizarin red staining; dark blue calcified nodules were found from cells after ALP staining.Conclusion BMSCs can be properly isolated by density gradient centrifugation combined with attached screening method.BMSCs differentiate to osteoblast after osteogenic induction and can be ideal seed cells for BTE.

关 键 词：骨髓 间充质干细胞 兔 分离 成骨分化 

分 类 号：R318[医药卫生—生物医学工程]