摘要
背景:胶质细胞神经营养因子在诱导骨髓间充质干细胞体外定向分化及促进神经元轴突再生过程中起到重要作用。目的:观察过表达胶质细胞神经营养因子基因诱导骨髓间充质干细胞向神经元样细胞分化情况,检测分化后细胞突触功能及Wnt信号通路组分表达,初步探索骨髓间充质干细胞向成熟神经元分化机制。方法:分离培养大鼠骨髓间充质干细胞,分为重组腺病毒载胶质细胞神经营养因子基因转染组(Ad-GDNF-BMSCs)、腺病毒转染对照组(Ad-BMSCs)及未转染对照组。Q-PCR检测各组骨髓间充质干细胞中胶质细胞神经营养因子基因相对表达量,免疫荧光技术检测各组细胞中β-catenin、cyclin D1、NeuN及MAP-2表达。高K+刺激诱导分化后细胞去极化反应,FM4-64标记分化后细胞突触囊泡活动。结果与结论:①腺病毒载目的基因转染对骨髓间充质干细胞增殖无显著负面影响,转染后可有效促进骨髓间充质干细胞持续、高水平表达内源性胶质细胞神经营养因子基因;②在胶质细胞神经营养因子基因诱导作用下体外培养3 d,骨髓间充质干细胞可表达神经元特异性蛋白NeuN,且在细胞胞质中检测到β-catenin蛋白表达;体外培养7 d后,骨髓间充质干细胞表达成熟神经元标记蛋白MAP-2,细胞胞体皱缩明显,胞体周围出现神经元轴突样结构,并在细胞胞质中检测到β-catenin、胞核中检测到cyclin D1表达,而Ad-BMSCs组及未转染对照组未见NeuN、MAP-2、β-catenin、cyclin D1表达且细胞仍维持梭形形态;③体外培养11 d后,Ad-GDNF-BMSCs组细胞呈现典型的神经元突起或轴突并相互连接成网状结构,可被FM4-64标记显示红色荧光,给予高K+刺激诱发细胞产生动作电位后轴突发生突触囊泡活动,可见FM4-64红色荧光逐渐衰减,同条件下Ad-BMSCs转染组及未转染组细胞未见FM4-64荧光标记的突触囊泡活动;④结果表明,胶质细胞神经营养因子持续诱
BACKGROUND:Glial cell line derived neurotrophic factor(GDNF)plays an important role in inducing the differentiation of bone marrow mesenchymal stem cells(BMSCs)in vitro and promoting the regeneration of neuron axons.OBJECTIVE:To observe BMSCs differentiation induced by over-expression of GDNF gene,detect synaptic function of cells and expression of Wnt signaling pathway components after differentiation,and preliminarily explore the mechanism of BMSCs differentiation into mature neurons.METHODS:Rat BMSCs were isolated and cultured,and further divided into recombinant adenovirus-containing GDNF gene transfection group(Ad-GDNFBMSCs),adenovirus transfection control group(Ad-BMSCs),and untransfected control group.The relative expression of GDNF gene in BMSCs of each group was detected by Q-PCR,and the expression ofβ-catenin,cyclin D1,NeuN and MAP-2 was detected by immunofluorescence technology in each group.High K+stimulation induced cell depolarization response after differentiation,and FM4-64 marks synaptic vesicle activity of differentiated cells.RESULTS AND CONCLUSION:(1)The adenovirus-transfected gene had no significant negative effect on the proliferation of BMSCs.BMSCs could express endogenous GDNF gene continuously and at high levels.(2)Under the induction of GDNF gene,BMSCs could express neuron-specific protein NeuN after 3 days cultivation in vitro.The expression ofβ-catenin protein also could be detected in the cytoplasm of cells.After 7 days cultivation in vitro culture,BMSCs expressed the mature neuronal marker protein MAP-2,and the cell body contracted significantly.Neuron axon-like structures appeared around the cell body.Moreover,β-catenin and cyclin D1 were respectively detected in the cell cytoplasm and the nucleus,while the expression levels of NeuN,MAP-2,β-catenin,and cyclin D1 were not observed in Ad-BMSCs and untransfected control groups,and the cells remained spindle-shaped.(3)After 11 days of in vitro culture,the cells in the Ad-GDNF-BMSCs group showed typical neuronal processes or axons an
作者
朱雪芬
黄成
丁健
戴永平
刘元兵
乐礼祥
王亮亮
杨建东
Zhu Xuefen;Huang Cheng;Ding Jian;Dai Yongping;Liu Yuanbing;Le Lixiang;Wang Liangliang;Yang Jiandong(Rugao People’s Hospital,Rugao 226500,Jiangsu Province,China;Department of Spine Surgery,Northern Jiangsu People’s Hospital,Yangzhou 225000,Jiangsu Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2021年第7期1019-1025,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金面上项目(81071466),项目负责人:杨建东
扬州市十三五科教兴卫领军人才计划资助(扬卫科教[2018]6号),项目负责人:杨建东
南通市科技局科研基金项目(GJZ16044),项目负责人:黄成
如皋市科研计划项目(SRG(15)3015),项目负责人:黄成。
关键词
干细胞
骨髓间充质干细胞
神经元
因子
突触
轴突
通路
stem cells
bone marrow mesenchymal stem cells
neurons
factors
synapses
axons
pathways
