人脐带间充质干细胞体外分化成骨细胞的研究

目的探讨人脐带源间充质干细胞(HUC-MSCs)定向诱导分化为成骨细胞的能力。方法人脐带经胶原酶和胰蛋白酶消化后于DMEM/F12培养基中培养,通过传代作纯化和扩增;采用倒置显微镜及电镜观察细胞形态,细胞计数绘制细胞生长曲线,流式细胞仪检测细胞免疫表型及细胞周期;以含有成骨细胞诱导剂(地塞米松、β-甘油磷酸钠、抗坏血酸)的DMEM/F12培养基对传至第3代的细胞定向诱导分化为成骨细胞,并对诱导后细胞作成骨细胞检测。结果经酶消化后,细胞贴壁生长,主要呈"成纤维样",但体积较大、形状不规则、细胞质突起多,细胞核大而疏松,核仁明显;第1、3、5、7代细胞生长曲线基本相同;细胞表面表达CD105、CD90、CD44、CD29及CD13,不表达CD34、CD45、HLA-DR;培养至第4代的细胞约72.724%的细胞处G1期、S期的细胞仅占18.069%,第6代时G1期细胞约为83.875%、S期为9.606%左右;细胞经成骨细胞诱导分化后,细胞形态发生改变,线粒体明显增多,出现较多的基质小泡、髓样体和空泡状结构,碱性磷酸酶染色显示呈强阳性,茜苏红染色和Von-Kossa染色显示细胞有钙盐沉积并形成钙结节。结论人脐带中分离及培养扩增细胞具有MSCs的基本特性,具有分化为成骨细胞的能力。

Study of mesenchymal stem cells from the human umbilical cord induced into osteoblasts in vitro

Objective To isolate mesenchymal stem cells(MSCs) from the human umbilical cord,identify their biological characteristics in vitro,and explore the possibility of differentiation into osteogenic cells.Methods Mesenchymal stem cells were separated from the umbilical cord with collagenase type Ⅳ and trypsin digestion to obtain the adherent cells in vitro.The cell morphology was observed by inverted microscopy and electron microscopy.Growth curves of cells were drawn.The expression of surface antigens and the cell cycle of MSCs were detected by f1ow cytometry.At the third passage,MSCs were induced to differentiate into osteoblasts with conditioned culture medium(dexamethasone,β-sodium glycerophosphate,ascorbic acid,bFGF and TGF-β).Results The cells showed a fibroblast-like irregular appearance,many cytoplasmic processes,loose chromatin,and a large nucleus with obvious nucleoli.The cell growth curves of the 1st,3rd,5th and 7th generations were basically the same.Flow cytometry analysis showed that adhesive cells were all positive for MSC-related antigens,such as CD29,CD105,CD44,CD13 and CD90,but negative for CD34,CD45,and HLA-DR.The cell cycle showed that about 72.724% of the cells of P4 were in G1 phase and 18.069% were in S phase;about 83.875% of the cells of P6 were in G1 phase and 9.606% of the cells were in S phase.After inducing for 21 days,differentiated osteoblasts showed an increase in mitochondria,a lot of matrix vesicles,a myelinated-like body,and a polyvacuolus-like structure that appeared in the cytoplasm,which were observed by the optical microscope and their ultrastructural characteristics by the electron microscope.ALP staining was positive 14 d after co-culture.Alizarin red and Von-Kossa′s stain revealed heavy extracellular matrix deposition and calcium nodule formation,and the calcium nodules appeared 21 d after co-culture.Conclusion The cultured cells from the umbilical cord can be amplified easily and passaged in vitro with the biological characteristics of MSCs.HUCMSCs could be induced to differentiate into osteoblasts successfully.