成人骨髓基质干细胞体外培养及定向诱导分化为成骨细胞  

In vitro culture of human bone marrow-derived mesenchymal stem cells and induction into osteoblasts

作　　者：滕勇[1] 胡蕴玉[1] 安书杰[2] 戴先文[1] 赵黎[1] 白建萍[1] 李旭升[1] 吕荣[1] 

机构地区：[1]第四军医大学西京医院骨科,陕西西安710033 [2]第四军医大学研究生院,陕西西安710033

出　　处：《第四军医大学学报》2004年第16期1492-1495,共4页Journal of the Fourth Military Medical University

基　　金：军队"十五"重点课题 (0 1Z0 79)

摘　　要：目的 :建立成人骨髓基质干细胞 (MSCs)体外培养的方法 ,并探讨定向诱导分化为成骨细胞的途径 .方法 :抽取成人骨髓 ,Percoll密度梯度离心法进行体外培养 ,贴壁细胞传代 ,取第 3代细胞在培养基中添加成骨诱导剂地塞米松、β 甘油磷酸、维生素C ,培养 1 5d后 ,在倒置显微镜观察细胞形态 ,钙 -钴法染色检测碱性磷酸酶 (ALP)表达 ,免疫组化 (SABC法 )检测Ⅰ型胶原、骨钙素表达 ,茜素红染色检测钙结节形成 .结果 :Percoll密度梯度离心法培养可获得均一的MSCs;诱导分化的MSCs呈典型的成骨细胞形态 ;ALP染色阳性率可达81 %以上 ;Ⅰ型胶原、骨钙素表达阳性 ;茜素红染色见钙结节形成 .结论 :可以从成人骨髓中培养出MSCs,并可定向诱导分化为成骨细胞 。AIM: To build a method to culture bone marrow derived mesenchymal stem cells (MSCs) in vitro and to investigate the feasibility of inducing MSCs into osteoblasts. METHODS: Human MSCs were isolated from adult bone marrow and purified by percoll density gradient centrifugation and cultured in vitro . The MSCs attachment formed after 7-10 d and the MSCs of the passage 3 were chosen to induce into osteoblasts. Fifteen days later, the alkaline phosphatase assay was conducted using modified calcium cobalt staining method, type Ⅰ collagen and osteocalcin assay was conducted using immunohistochemistry and calcium node assay was done using alizarin red staining. RESULTS: Homogeneous MSCs were obtained by percoll density gradient centrifugation. The induced MSCs had typical appearance of osteoblasts. The rate of ALP expression was 81%, the expressions of collagen type Ⅰand osteocalcin were positive, and calcium nodes were seen. CONCLUSION: The MSCs can be separated from adult bone marrow and cultured in vitro , which can be induced into osteoblasts.

关 键 词：骨髓 干细胞 成年人 组织工程 骨生成/药物作用 

分 类 号：R322.71[医药卫生—人体解剖和组织胚胎学] R329.2[医药卫生—基础医学]