中国输血杂志2011，Vol.24Issue(5)：376-382,7.
人脐带间充质干细胞体外分化成骨细胞的研究
Study of mesenchymal stem cells from the human umbilical cord induced into osteoblasts in vitro
摘要
Abstract
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 flow 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 1 st ,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.关键词
间充质干细胞/人脐带/体外培养/细胞周期/生长曲线/成骨细胞Key words
Mesenchymal Stem Cells/ Culture in Vitro, Human Umbilical Cord/ Cell cycle/ Growth Curves/ Osteoblasts分类
医药卫生引用本文复制引用
刘忠,李素萍,杨宏友,王震,吕蓉,吴炳,方勤,李敏,吴学忠..人脐带间充质干细胞体外分化成骨细胞的研究[J].中国输血杂志,2011,24(5):376-382,7.基金项目
国家科技支撑计划(2007BAI39B02)、安徽省卫生厅重点科研项目(NO.07080703011) （2007BAI39B02）
