人脐带间充质干细胞经添加B27的无血清培养基诱导后分化成神经元样细胞  

Human umbilical cord mesenchymal stem cells differentiate into neuron-like cells after induction with B27-supplemented serum-free medium

作　　者：李云逸[1] 杨锦培 傅国 周攀 刘阳 李志忠[2] 焦根龙[1] LI Yunyi;YANG Jinpei;FU Guo;ZHOU Pan;LIU Yang;LI Zhizhong;JIAO Genlong(First Affiliated Hospital of Jinan University,Guangzhou 510632,China;Shenhe Hospital Affiliated to Jinan University,Heyuan 517000,China)

机构地区：[1]暨南大学附属第一医院,广东广州510632 [2]暨南大学附属深河医院,广东河源517000

出　　处：《南方医科大学学报》2020年第9期1340-1345,共6页Journal of Southern Medical University

基　　金：中国广东省医学科学研究基金会(A2017475);中央高校基础研究基金(21619419)。

摘　　要：目的评价人脐带间充质干细胞(HUCMSCs)经添加B27的无血清培养基诱导后向神经元样细胞分化的能力和效率。方法将第4代HUCMSCs分为4组:A组:血清培养基(SCM)处理14 d;B组:给予SCM+20 ng/mL神经生长因子(NGF)+10 ng/L碱性成纤维细胞生长因子(bFGF)治疗14 d;C组:无血清培养基(SFM)处理14 d;D组:SFM+20 ng/mL NGF+10 ng/mL bFGF处理。每3 d更换各组细胞培养液。倒置显微镜观察神经球生长情况,流式细胞术分析细胞标记物。巢蛋白(Nestin)、神经元特异性烯醇化酶(NSE)、人重肽神经丝蛋白(NEFH)、神经胶质纤维酸性蛋白(GFAP)通过qRT-PCR和Western blotting进行定量。结果在诱导前,HUCMSCs表现出丰富的间充质干细胞表面标志物(CD29、CD44和CD105分别为99.5%、49.6%和77.7%)的表达。在第7、10、14天观察神经元样细胞的形成,D组最优,其次是C、B、A组。qRT-PCR和Western blotting结果显示,A、B、C、D组Nestin和GFAP表达逐渐减少,NEFH和NSE表达逐渐增加,A组与其他3组的GFAP、NEFH、Nestin和NSE表达差异有统计学意义(P<0.05)。结论添加B27的SFM可以有效地将HUMSCs分化为神经元样细胞,添加细胞因子(NGF和bFGF)使分化效果更显著。Objective To evaluate the capacity and efficiency of human umbilical cord mesenchymal stem cells(HUCMSCs)to differentiate into neuron-like cells after induction with B27-supplemented serum-free medium.Methods HUCMSCs at passage 4 were cultured for 14 days with serum-containing medium(SCM)(group A),SCM supplemented with 20 ng/mL nerve growth factor(NGF)and 10 ng/mL basic fibroblast growth factor(bFGF)(group B),serum-free medium(SFM)(group C),or SFM supplemented with 20 ng/mL NGF and 10 ng/mL bFGF.The culture medium were changed every 3 days and the growth of the neurospheres was observed using an inverted microscope.The cell markers were analyzed with flow cytometry and the expressions of nestin,neuron-specific enolase(NSE),neurofilament heavy polypeptide(NEFH),and glial fibrillary acidic protein(GFAP)were quantified by quantitative real-time PCR(qRT-PCR)and Western blotting.Results Before induction,HUCMSCs expressed abundant mesenchymal stem cell surface markers including CD29(99.5%),CD44(49.6%)and CD105(77.7%).Neuron-like cells were observed in the cultures on days 7,10,and 14,and the cell differentiation was the best in group D,followed by groups C,B and A.In all the 4 groups,the cellular expressions of nestin and GFAP gradually lowered while those of NEFH and NSE increased progressively.The expressions of GFAP,NEFH,nestin and NSE were significantly different between group A and the other 3 groups(P<0.001 or 0.05).Conclusion B27-supplemented SFM effectively induces the differentiation of HUCMSCs into neuron-like cells,and the supplementation with cytokines(NGF and bFGF)strongly promotes the cell differentiation.

关 键 词：人脐带间充质干细胞 神经元 分化 无血清培养基 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]