慢病毒介导神经生长因子过表达诱导脐带间充质干细胞向神经元样分化  

Lentivirus-mediated overexpression of nerve growth factor induces differentiation of umbilical cord mesenchymal stem cells into neuron-like cell

作　　者：陈浩浩[1] 郑群[2] 王文倩 江文娇 王飞凤[2] 傅晓艳[1] 张宁[1] Chen Haohao;Zheng Qun;Wang Wenqian;Jiang Wenjiao;Wang Feifeng;Fu Xiaoyan;Zhang Ning(Medical Molecular Biology Laboratory,School of Medicine,Jinhua Polytechnic;Center of Reproductive Medicine,Jinhua people’s Hospital;Jinhua Sidanmu Stem Cell Biotechnology Limited Company,Jinhua 321000,china)

机构地区：[1]金华职业技术学院医学院分子生物学实验室 [2]金华市人民医院生殖医学中心 [3]金华市思丹姆干细胞生物科技有限公司,金华321000

出　　处：《中国组织化学与细胞化学杂志》2018年第5期422-427,共6页Chinese Journal of Histochemistry and Cytochemistry

基　　金：金华市科学技术研究计划(2017-4-069);浙江省公益技术应用研究计划(2015C37104)

摘　　要：目的探讨神经生长因子(NGF)过表达慢病毒转染脐带间充质干细胞(UMSCs)对细胞分化的影响。方法分离培养UMSCs,流式细胞术鉴定后利用慢病毒载体感染细胞,使其过表达NGF,72h后,通过免疫荧光检测NGF和GFP的表达,Western blot检测NGF蛋白表达水平,ELISA法检测细胞上清液NGF的含量,qRT-PCR检测相关神经因子基因的表达。结果流式细胞术显示细胞表面CD105、CD90、CD73阳性而CD34、CD45、CD19和CD14阴性,鉴定为脐带间充质干细胞。慢病毒转染后细胞NGF和GFP阳性,细胞内NGF和培养液中b-NGF含量都显著性增加,NGF、nestin、GFAP、MAP2及tubulin等mRNA的表达显著性增加。结论神经生长因子过表达慢病毒转染脐带间充质干细胞会促使细胞向神经元样细胞分化,可应用于后续研究。Objective To investigate the effect of nerve growth factor(NGF)overexpression via lentivirus transfection on the differentiation of umbilical cord mesenchymal stem cells(UMSCs).Methods UMSCs were isolated and cultured,then identified by flow cytometry,and then transfected with lentiviral vectors to overexpress NGF.After 72 hours,the expression of NGF and GFP was detected by immunofluorescence,and the expression of NGF protein was detected by Western blot.In the cell supernatant,the content of NGF was detected by ELISA and the expression of related neural factor genes was detected by qRT-PCR.Results Flow cytometry results showed that cells were CD90,CD73 and CD105 positive,CD34,CD45,CD19 and CD14 negative on the surface,which identified the cells as UMSCs.After lentivirus transfection,it was found that the cells were positive for NGF and GFP,both content of intracellular NGF and supernatant NGF increased significantly,furthermore mRNA expression of NGF,nestin,GFAP,MAP2,and tubulin was increased significantly.Conclusion Nerve growth factor overexpression via lentiviral transfection of umbilical cord mesenchymal stem cells promotes differentiation of cells into neuron-like cells,which can be used in subsequent studies.

关 键 词：神经生长因子 脐带间充质干细胞 神经元分化 

分 类 号：R329.1[医药卫生—人体解剖和组织胚胎学]