枸杞多糖诱导人脐血间充质干细胞向神经元样细胞分化的实验研究

Study of Differentiation of Human Umbilical Cord Blood Mesenchymal Stem Cells Into Neuron-like Cells Induced by Lycium Barbarum Polysaccharine

目的:探讨枸杞多糖诱导人脐血间充质干细胞(MSCs)向神经元样细胞分化的可行性及其机制。方法:无菌条件下收集正常足月儿的脐带血,经肝素抗凝,用相对密度1.077的淋巴细胞分离液分离脐血的单个核细胞,用低糖DMEM培养基进行培养和纯化扩增。选取第3代细胞进行诱导实验,当传代细胞长满瓶底的80%以上时,先用含15%FBS和10ng/ml bFGF的DMEM完全培养基预诱导24小时,然后用不含血清含1g/L枸杞多糖的DMEM培养基诱导,光镜下观察细胞形态,用免疫组化技术检测细胞Nestin和NF的表达。结果:预诱导后MSCs没有变化,而经枸杞多糖诱导4h后细胞即出现形态学上的改变,细胞变成不规则形,立体感增强,从胞体伸出突起。免疫组化检测显示,细胞Nestin、NF呈阳性。结论:人脐血间充质干细胞经枸杞多糖诱导可转化为神经元样细胞,其诱导机制可能与枸杞多糖的抗氧化作用有关。

Objective： To study the differentiation possibility and mechanism of human umbilical cord blood mesenchymal stem cells （MSCs） into neuron-like cells induced by lycium barbarum polysaccharine （LBP）. Methods： Human umbilical cord blood was collected from mature neonates. All samples were obtained sterilely with 20 U/ml heparin, The cord mononuclear cells were isolated with lymphocyte separation medium （density 1.077g/ml）, then purified and expanded with slight sugar DMEM medium, The third passage of the expanded MSCs were pretreated with pre-induction medium consisting of DMEM with 150% FBS and 10ng/ml bFGF for 24 hours, then induced with induction medium consisting of DMEM without serum but 1g/L lycium barbarum polysaccharine. The morphological changes of the cells were observed under phase contrast microscope, The neuron specific markers, nestin and neurofilament （NF）, were evaluated by indirect immunocyto-chemistry staining. Results： After induction for 4 hours, some showed morphological changes obviously. Twenty-four hours later, the differentiated cells showed typical neuronal morphology. The expression of nesfin and NF were positive. Conclusion： Lycium barbarum polysaccharine may induce the human umbilical cord blood MSCs into neuron-like cells . The mechanism of such induction may be related with its anti-oxidation characteristics.