氯化锂体外诱导人脐血间充质干细胞向神经细胞分化的实验研究

目的 探讨氯化锂体外诱导人脐血间充质干细胞(MSCs)向神经细胞分化的可行性.方法 无菌条件下收集正常足月儿的脐带血,肝素抗凝,密度梯度离心法分离人脐血单个核细胞,贴壁法纯化,用含15%FBS的低糖DMEM培养基进行扩增培养,流式细胞仪检测表面抗原.取3代的脐血MSCs进行诱导,A组:用含15%FBS和20 ng/ml bFGF的DMEM完全培养基预诱导24 h,3 mol/L LiCI的DMEM培养基继续诱导6 d:B组:含3 mol/L LiCI的DMEM培养基诱导7 d;C组:含15%FBS的DMEM培养基正常培养7 d.光镜下观察细胞形态,用免疫组化技术检测细胞NSE、MAP2及GFAP的表达.结果 A组与B组诱导3 d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.免疫组织化学和免疫荧光方法鉴定显示,诱导后的细胞能表达神经元特异性标志NSE和MAP2,阳性表达率A组分别为(73.6±7.8)%,(75.5±8.5)%]明显高于B组分别为(31.0±4.3)%、(33.5±5.O)%],而星形胶质细胞特异性标志GFAP阳性细胞较少,A、B、C三组阳性表达率分别为(4.7 ±3.3)%、(5.1±4.6)%、(8.5±3.2)%.结论 LiCI联合生长因子bFGF体外可诱导人脐血MSCs分化为神经元样细胞.

The experimental study on differentiation of human umbilical cord blood mesenchymal stem cells into neural cells induced by lithium chloride

Objective To study the possibility of inducing human umbilical cord blood mesenchymal stem cells(MSCs) to differentiate into neuron-like cells by lithium chloride(LiCl) in vitro.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 1.077 g/ml), then purified by wall sticking screening and expanded with slight sugar DIEM containing 15% FBS.The third passage of the expanded MSCs were pre-inducted with DIEM containing 15% FBS and 20 ng/ml bFGF for 24 hours, then induced with DIEM without serum but 3 mol/L LiCl for 6 days in group A.The MSCs were induced with DIEM containing 3 mol/L Licl for 7 days in group B.The MSCs were normally cultured with DIEM containing 15% FBS in group C.The morphological changes of the cells were observed under phase contrast microscope.The neuron specific markers containing neuron specific enolase(NSE), microtubule associated protein2(MAP2) and glial fibrillary acid protein(GFAP) were evaluated by indirect immunocyto-chemistry staining.Results After inducted for 3 days, morphological changes were observed obviously in group A and B.6 days later, the differentiated cells showed typical neuronal morphology.The expression of NSE and MAP2 were positive for the majority cells in group A and B, and that of group A(73.6 ± 7.8)%, 75.5 ± 8.5)% respectively]were obviously higher than group B(31.0 ± 4.3)%,(33.5 ± 5.0)% respectively], few expressed GFAP in both groups.Conclusion The combination of LiCl and growth factor may induce the human umbilical cord blood MSCs into neuron-like cells in vitro.