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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 ,可用作临床骨组织工程种子细胞
OBJECTIVE: To establish a method for culturing adult bone marrow stromal stem cells (MSCs) in vitro and to explore ways of differentiating into osteoblasts.Methods: Adult bone marrow was harvested and percoll density gradient centrifugation was used to culture in vitro and adherent cells were passaged. Cells were cultured in culture medium containing dexamethasone, β-glycerophosphate and vitamin C for 15 days. After culturing for 15 days, the morphology of cells was observed under an inverted microscope. The expression of alkaline phosphatase (ALP) was detected by calcium-cobalt staining. Type Ⅰ collagen was detected by SABC method, osteocalcin expression and alizarin red staining were used to detect the formation of calcium nodules.Results: Percoll density gradient centrifugation was used to obtain uniform MSCs.The differentiated MSCs showed typical osteoblasts The positive rate of ALP staining was over 81%, the expression of type Ⅰ collagen and osteocalcin was positive, and the alizarin red staining showed the formation of calcium nodules.Conclusion: MSCs can be cultured from adult bone marrow and induced to differentiate into Bone cells, can be used as clinical bone tissue engineered seed cells