兔骨髓间充质干细胞分离培养及体外诱导分化为成骨细胞  

Isolation and Culturing of Rabbit Bone Marrow Mesenchymal Stem Cells and Its Induced Differentiation Into Osteoblasts in vitro

作　　者：周永春[1] 鲁超[2] 宋宗让[1] 孟羿彬[1] 郝定均[1] 

机构地区：[1]西安交通大学医学院附属红会医院脊柱外科,陕西西安710054 [2]西安交通大学医学院附属红会医院关节外科,陕西西安710054

出　　处：《医学临床研究》2015年第4期702-706,共5页Journal of Clinical Research

摘　　要：【目的】建立兔骨髓间充质干细胞（MSCs）的分离、纯化及鉴定方法，观察体外成骨潜能。【方法】应用密度梯度离心联合贴壁筛选的方法，体外分离培养兔骨髓来源的M SCs ，培养过程中倒置显微镜下观察其生物学特性，流式细胞仪检测细胞表型，所得的细胞进行成骨诱导分化后进行碱性磷酸酶（alkaline phos‐phatase ，ALP）染色及茜素红染色鉴定，并对成骨分化指标进行检测。【结果】原代分离培养的贴壁细胞呈长梭形，漩涡状排列。传代后增殖迅速，细胞为单一的梭形，排列更加有序。培养的细胞CD44呈阳性表达，而CD34呈阴性表达。成骨诱导后ALP染色和茜素红染色均呈阳性。诱导组成骨分化后成骨标志物含量较对照组明显要高。【结论】密度梯度离心结合贴壁的方法可以获得高纯度MSCs ，增殖旺盛，体外具有成骨潜能。[Objective] To establish the experimental method for isolating and culturing rabbit bone‐de‐rived mesenchymal stem cells （MSCs） and examine their osteogenic potentials in vitro .[Methods]Mesenchy‐mal stem cells （MSCs） were isolated and purified by density gradient centrifugation plus attachment culture . The biological characteristics of MSCs were observed under inverted microscope .Cell surface markers were as‐sessed by flow cytometry and MSCs identified with alkaline phosphatase （ALP） and Alizarin red staining after osteogenic differentiation .Finally the markers of osteogenic differentiation were detected .[Results] The ad‐hered cells by primary culture were spindle and whirlpool‐shaped .After passage ,the cells became spindle‐shaped and distributed orderly .Cultured cells were positive for CD44 ,but negative for CD34 .After osteogenic induction ,ALP and Alizarin red stains were positive .After induced osteogenic differentiation ,the content of osteogenic markers in osteogenic differentiation group was significantly higher than that of control group .[Conclusion] Highly purified MSCs may be obtained by density gradient separation and screening adherence . And MSCs possess osteogenic potential in vitro .

关 键 词：间质干细胞 细胞分离 细胞分化 成骨细胞 兔 

分 类 号：R329.25[医药卫生—人体解剖和组织胚胎学]