摘要
目的探讨应用无血清培养体系分离培养脐带间充质干细胞,明确其成骨生物学特性,为临床应用奠定实验基础。方法在无血清干细胞培养体系下,利用组织块贴壁培养法分离培养,扩增脐带间充质干细胞,在倒置显微镜进行形态学观察,流式细胞检测鉴定其表面标记CD34、CD44、CD90及CD45的表达,成骨诱导液培养2～3周后观察其细胞形态学变化,茜素红染色鉴定其成骨情况。结果脐带组织块接种后第1次更换培养液12h后,有少量梭形的细胞从脐带组织边缘爬出,培养5～6d左右成单层状生长,12d左右梭形状细胞呈现复层生长趋势,细胞表面标记CD44、CD90均呈阳性为间充干细胞来源,CD34、CD45均呈阴性为非造血干细胞来源;细胞经成骨诱导2～3周后具有良好的分化成骨能力。结论利用无血清的人间充质干细胞专用培养基培养体系,脐带组织培养法能够分离培养出大量的脐带间充质干细胞,避免了培养中异种蛋白的混入而降低临床排异反应。
Objective To identify the isolated and purified mesenchymal stem cells（UC-MSCs） derived from umbilical cord by serum free culture medium in vitro in order to know more about its role in osteogenesis which may provide basic data for applying potentially clinical treatment in the future.Methods Nubbles of umbilical cord tissue were cultured with serum free culture medium.UC-MSCs were observed under an inverted microscope.Grown UC-MSCs were isolated and purified from umbilical cord tissue cultures without any serum.UC-MSCs＇ markers CD34、CD44、CD90 and CD45 were detected by flow cytometry.The cells were induced by bone induction culture medium for 2～3 weeks and then differentiated to osteoblast-like cells.The morphological changes on cell surface were monitored during cell differentiation,and osteoblast-like cells were further confirmed by alizarin red staining.Results A small amount of spindle cells were found to scrawl out from umbilical cord tissue blocks after having been cultured in serum free medium for 12 hours.A monolayer of cells was confluent on the plates on 5-6th day,and then stratified into multi-layers after 12th day.The cell surface markers were positive for CD44,CD90,and negative for CD34,CD45 indicating that the cells were originally from mesenchymal stem cells but not from hematopoietic ones.UC-MSCs had been differentiated into osteoblast-like cells after osteoblast induction for 2-3 weeks.Conclusion Using human mesenchymal stem cell-specific medium culture system without any serum,a large amount of UC-MSCs can effectively be isolated and harvested.The cells can be induced into osteoblasts and avoid any heterogeneous proteins interfusion so as to reduce clinical applications.
出处
《同济大学学报（医学版）》
CAS
2010年第5期21-24,共4页
Journal of Tongji University(Medical Science)
基金
上海市科委浦江人才计划资助项目(07PJ00363)
