人脐血间充质干细胞的体外培养及生物学特性

目的:建立一种体外纯化增殖人脐血源性间充质干细胞(MSCs)的实验方法,并观察其生物学特性。方法:从正常分娩的足月胎儿脐血中分离出MSCs进行原代培养,培养瓶预先用自体血浆包被处理。72-96 h半量更换培养液,弃去未贴壁细胞,以后每3-5 d半量换液1次。待细胞达到80%-90%融合时,按1∶2的比例进行传代。倒置或相差显微镜下每日观察原代和传代细胞的生长情况及形态学特征。取第3代脐血MSCs用流式细胞仪检测细胞周期和细胞表面抗原CD29、CD34、CD44、CD45的表达。结果:脐血经分离、培养5-7 d后有间充质样细胞贴壁,3-4周后,细胞生长达80%-90%融合,形态学类似骨髓源性MSCs。连续传至5代以后,增殖能力未见明显减弱。脐血MSCs的大部分细胞(>90%)处于G0/G1期,且均一稳定地表达间充质干细胞表面抗原CD29、CD44,但不表达CD34、CD45。结论:脐带血内含有MSCs,并可在体外纯化增殖,该技术可为实验研究和临床应用提供足够的干细胞来源。

Culture and Identification of Mesenchymal Stem Cells Derived from Umbilical Cord Blood

Objective: To establish a method of purification and culturing of mesenchymal stem cells(MSCs) derived from umbilical cord blood and to investigate their biological characteristics.Methods: MSCs were obtained from umbilical cord blood of term deliveries after the mothers signed a donation form.The culture bottles were pretreated coating with self plasma.After 72-96 hours of primary culture,one half of the medium was renewed and non-adherent cells were removed.The medium was renewed by half in every 3-5 days until MSCs reached 80%-90% fusion,then they were subcultured with a propotion of 1∶2.After the third passage,the cells were analyzed with flow cytometry.The following monocloned antibodies as CD29,CD34,CD44 and CD45 were used.The growth cycle and morphological changes of MSCs were monitored by phase contrast microscopy.Results: A cell line derived from umbilical cord blood was acquired which possessed morphological and immunophenotypical characteristics similar to those of MSCs isolated from bone marrow.The potential of proliferation remained unchanging over five passages when the cells were cultured and maintained at low differentiation.By the flow cytometric analysis,CD29 and CD44 were expressed on MSCs while no expression of CD34 and CD45 were found.Conclusion: The presence of MSCs in umbilical cord blood of term infants has been demonstrated,and MSCs can be purified and cultured in vitro for the further studies of the stem cells.