脐血间充质干细胞分化为神经样细胞的诱导因素的比较及优化  

Comparison and optimization of in vitro induction conditions of human umbilical cord blood-derived mesenchymal stem cells differentiated into neuron-like cells

作　　者：陈瑶[1] 张馨月[1] 陆玉霞[1] 徐文慧[1] 徐廷云[1] 褚静英[1] 

机构地区：[1]苏州卫生职业技术学院检验教研室,江苏苏州215009

出　　处：《临床检验杂志》2016年第9期669-673,共5页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省卫生职业技术教育立项课题(J201302)

摘　　要：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 explore the optimal conditions of human umbilical cord blood-derived mesenchymal stem cells （hUCB- MSCs） differentiated into neuron-like cells. Methods The hUCB-MSCs were obtained by density gradient centrifugation, and their immunophenotypes such as CD34, CD29 and CD44 were detected by flow cytometry （FCM）. The hUCB-MSCs were induced by 5 kinds of combinations with different inducing factors, including epidermal growth factor （ EGF ） ＋ basic fibroblast growth factor （ bFGF）, ratinoic acid （RA） ＋ EGF ＋ bFGF, RA ＋ brain-derived neurotrophic factor （BDNF） ＋ bFGF, tanshinol ＋ EGF ＋ bFGF, and β-mercaptoethanol （[3-ME） ＋ dimethyl sulfoxide （DMSO） ＋ butylhydroxy anisole （BHA）, respectively. The expression levels of neu- ron nucleoantigen （NeuN）,β-Tubulin-lll and glial fibrillary acidic protein （GFAP） in hUCB-MSCs were detected by the immunofluo- rescence method before and after induction. Results There were strong expressions of CD29 （96.2 ± 3.2） % and CD44 （97.1± 2.4） % and weak expression of CD34 （4.1± 2.1 ）% in hUCB-MSCs, hUCB-MSCs induced by 5 combinations with different inducing factors displayed neuron-like morphological changes. Immunofluorescence results showed that the induced hUCB-MSCs expressed NeuN and β-Tubulin- ]H highly, but GFAP lowly. The cell rates with positive NeuN （ 42.7 ± 3.1 ） % and β-Tubulin- Ⅲ （ 55.4 ± 2.2 ） % were the lowest in the EGF ＋ bFGF group, and the highest （78.9 ± 4.4） % and （79.6 ± 3.3 ） % in the tanshinol ＋ EGF ＋ bFGF group （P 〈 0.05 ）. Conclusion hUCB-MSCs may differentiate into neurons and neurogliocytes after induced by 5 combinations with differ- ent inducing factors, among which the combination of tanshinoh EGF and bFGF has the best induction.

关 键 词：脐血间充质干细胞 神经样细胞 表皮生长因子 碱性成纤维细胞生长因子 

分 类 号：Q291[生物学—细胞生物学]