脐血间充质干细胞体外扩增和向神经元样细胞的定向诱导*

背景：骨髓间充质干细胞存在取材困难、供体有限、可能病毒污染等缺陷，限制了其临床应用，而目前已证实间充质干细胞不仅存在于骨髓，还存在于外周血，尤其是脐血。 目的：探讨人脐血间充质干细胞的体外分离、扩增纯化方法及其神经分化潜能。 设计、时间及地点：应用研究，体外细胞学观察，于2005-10/2007-10在南方医科大学神经生物学教研室完成。 材料：新鲜脐血来自南方医科大学南方医院足月剖宫产新生儿脐带，取脐血前征得新生儿监护人的知情同意。 方法：无菌条件下收集脐血，去除红细胞，采用低糖DMEM/F12进行培养；取扩增第3或4代的人脐血间充质干细胞，培养基中添加碱性成纤维细胞生长因子、表皮生长因子和全反式维甲酸。 主要观察指标：用流式细胞仪检测贴壁细胞的表面标志；用免疫组化法检测诱导后细胞神经元特异性烯醇化酶、巢蛋白、神经元特异性核内抗原和神经丝蛋白表达。 结果：人脐血间充质干细胞强表达CD13、CD 29、CD44和CD105，弱表达CD106，不表达CD34、CD11a、CD14、CD33和CD45。培养基添加神经营养因子诱导后的细胞呈现典型的神经元样表型，诱导后高表达巢蛋白、神经元特异性烯醇化酶、神经元特异性核内抗原和神经丝蛋白。 结论：人脐血富含间充质干细胞，分离培养后于培养基添加神经营养因子能够分化为神经元样细胞。

Amplification and directional differentiation in vitro of human umbilical cord blood derived mesenchymal stem cells into neuron-like cells

BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are difficult to get for limited donor and virus polluted, which limit clinical application. Some more report found that BMSCs exist in peripheral blood and cord blood. OBJECTIVE: To investigate in vitro isolation, purification, amplification and differentiation towards neuron-like cells of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs). DESIGN, TIME AND SETTING: Exploratory development, in vitro cytology observation was performed at the Institute of Neurobiology, Southern Medical University from October 2005 to October 2007. MATERIALS: Fresh umbilical cord blood was obtained from full-term uterine-incision delivery neonates at the Nanfang Hospital of Southern Medical University after the informed constant was obtained from guardians of neonates. METHODS: Umbilical cord blood was collected in sterile condition, after which erythrocyte was destroyed. The collected cell clump was cultivated with low-glucose DMEM/F12. The third to fourth passages of UCB-MSCs were incubated in culture medium containing basic fibroblast growth factor, epidermal growth factor and all-trans retinoic acid. MAIN OUTCOME MEASURES: Surface markers of adhered cells were detected using flow cytometry. Expressions of neuronspecific enolase, nestin, NeuN and neurofilament protein about induced cell were detected by immunohistochemistry. RESULTS: UCB-MSCs strongly expressed CD13, CD29, CD44 and CD105, weakly expressed CD106, and did not express CD34, CD11a, CD14, CD33 or CD45. After added neurotrophic factor into medium, the majority cells assumed the morphology of neuron-like cells, and which strongly expressed nestin, neuronspecific enolase, NeuN and neurofilament protein. CONCLUSION: Mesenchymal stem cells are rich in human umbilical cord blood. After isolation and culture, mesenchymal stem cells incubated in medium with neurotrophic factor can differentiate into neurons-like cells.