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

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.