【摘要】 目的探讨体外分离、培养山羊骨髓间充质干细胞(BMSCs),并将其诱导分化为软骨细胞表型,明确其向软骨细胞分化的能力。方法抽取10月龄健康中国青山羊骨髓,贴壁法培养BMSCs,体外培养至第4代时进行流式细胞术鉴定,并加入成软骨诱导培养基,其成分包括10%FBS高糖DMEM培养基、6.25μg/ml胰岛素、6.25μg/ml转铁蛋白、50μmol/ml抗坏血酸、100nmol/L地塞米松及10ng/ml TGF-β1。分别于0、1、2、4周行细胞化学染色、逆转录-聚合酶链反应(RT-PCR)和蛋白印迹试验(Western blot)检测II型胶原和Aggrecan的表达。结果山羊BMSCs生长良好,传至第4代时细胞纯度较高。加入诱导培养基后,山羊BMSCs在1、2、4周均可看到软骨细胞表型的表达,并且随着时间的延长,其II型胶原及Aggrecan基因和蛋白的表达量逐渐增加,2周时即可达到较好的诱导效果。结论本实验采取一种简易的方法分离、培养并向软骨细胞方向诱导分化山羊BMSCs,证实其具有分化为软骨细胞的潜能,为山羊用于骨软骨组织工程的体内研究提供了实验基础。更多还原

【Abstract】 Objective To explore the isolation,in vitro culture and chondrogenic differentiation of goat bone marrow mesenchymal stem cells(BMSCs).Methods Bone marrow was harvested from a 10-month-old Chinese goat for adherent culture of the BMSCs in vitro.Flow cytometry was performed to detect the cell surface markers of the BMSCs of the fourth generation.The induction medium(containing 10% fetal bovine serum,high-glucose DMEM,6.25 μg/ml insulin,6.25 μg/ml transferrin,50 μmol/ml vitamin C,100 nmol/L DXM and 10 ng/ml transforming growth factor-β1) was then applied for chondrogenic differentiation.Cytochemical staining,RT-PCR and Western blotting were performed to detect the expressions of type II collagen and aggrecan in the cells at the time points of 0,1,2 and 4 weeks.Results The goat BMSCs grow well in vitro with a high purity in the fourth generation.The expression of chondrocyte phenotypes were observed at 1,2 and 4 weeks,which became more obvious as the culture prolonged.The mRNA and protein expression of type II collagen and aggrecan in the BMSCs increased obvious after the induction and had reached a satisfactory level by 2 weeks.Conclusion Goat BMSCs have the potential to differentiate into chondrocytes in vitro,and the results of this study provide the experimental basis for application of goat BMSCs in bone and cartilage tissue engineering in vivo.更多还原