论文部分内容阅读
目的探讨诱导人脐血间充质干细胞(h UCB-MSCs)向神经样细胞分化的最优条件。方法采用密度梯度离心法获得MSCs;流式细胞术分析h UCB-MSCs CD34、CD29、CD44等免疫表型;用5种不同诱导因子进行诱导:(1)表皮生长因子(EGF)+碱性成纤维细胞生长因子(b FGF);(2)维甲酸(RA)+EGF+b FGF;(3)RA+脑源性神经营养因子(BDNF)+b FGF;(4)丹参素+EGF+b FGF;(5)β-巯基乙醇(β-ME)+二甲基亚砜(DMSO)+丁羟基茴香醚(BHA);并用免疫荧光法检测诱导前后h UCB-MSCs神经元核抗原(Neu N)、β-微管蛋白-Ⅲ(β-Tubulin-Ⅲ)和神经胶质纤维酸性蛋白(GFAP)的表达水平。结果h UCB-MSCs中CD29[(96.2±3.2)%]、CD44[(97.1±2.4)%]呈强阳性,CD34呈弱阳性[(4.1±2.1)%]。h UCB-MSCs经5种方法诱导后均表现出类似神经元样细胞的形态改变。免疫荧光化学法结果显示,诱导后的细胞高表达Neu N和β-Tubulin-Ⅲ,低表达GFAP。EGF+b FGF诱导组Neu N、β-Tubulin-Ⅲ阳性细胞[(42.7±3.1)%,(55.4±2.2)%]在5个诱导组中表达最低(P<0.05);丹参素+EGF+b FGF诱导组(78.9±4.4%,79.6±3.3%)表达水平最高(P<0.05)。结论 h UCB-MSCs经不同诱导条件均能够向神经元和神经胶质细胞分化,丹参素+EGF+b FGF诱导法诱导效果最好。
Objective To investigate the optimal conditions for differentiation of human umbilical cord blood mesenchymal stem cells (h UCB-MSCs) into neuron-like cells. Methods MSCs were obtained by density gradient centrifugation. The immunophenotypes of hUCB-MSCs such as CD34, CD29 and CD44 were analyzed by flow cytometry. Five different inducing factors were used to induce MSCs. (1) Epidermal growth factor (EGF) (2) RA / EGF + b FGF; 3) RA + BDNF + b FGF; 4) Danshensu + EGF + b FGF (5) β-mercaptoethanol (DMSO) + butylated hydroxyanisole (BHA); and the levels of Neu N in h UCB-MSCs neurons before and after induction were detected by immunofluorescence staining. , Β-Tubulin-Ⅲ and glial fibrillary acidic protein (GFAP). Results CD29 [(96.2 ± 3.2)%] and CD44 [(97.1 ± 2.4)%] h UCB-MSCs were strongly positive and CD34 was weakly positive [(4.1 ± 2.1)%]. h UCB-MSCs showed similar morphological changes of neuron-like cells after being induced by five different methods. The results of immunofluorescence assay showed that the induced cells highly expressed Neu N and β-Tubulin-Ⅲ, with low expression of GFAP. The expression of Neu N, β-Tubulin-Ⅲ positive cells in EGF + b FGF-induced group was the lowest (P <0.05), while the expression of Danshensu + EGF + b FGF induced group (78.9 ± 4.4%, 79.6 ± 3.3%) had the highest expression level (P <0.05). Conclusion h UCB-MSCs can differentiate into neurons and glial cells under different induction conditions, and the induction effect of Danshensu + EGF + b FGF is the best.