文章摘要
孟 明,陈兰兰,陈伟贤,万 琪.神经营养因子相关基因在骨髓基质细胞分化为神经元中的表达[J].南京医科大学学报,2008,28(5):640~645
神经营养因子相关基因在骨髓基质细胞分化为神经元中的表达
Gene expression pattern of neurotrophins and their receptors during neuronal differentiation of rat bone marrow stromal cells
投稿时间:2007-12-10  
DOI:10.7655
中文关键词: 骨髓基质细胞  分化  神经营养因子  受体
英文关键词: bone marrow stromal cells  differentiation  neurotrophin  receptor
基金项目:
作者单位
孟 明 南京医科大学第一附属医院神经内科,江苏 南京 210029 
陈兰兰  
陈伟贤  
万 琪  
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中文摘要:
      目的:体外诱导骨髓基质细胞分化为神经细胞,并观察诱导分化前后神经营养因子及其受体相关基因的表达变化,以探索骨髓基质细胞诱导分化为神经细胞的可能机制?方法:体外培养经Percoll(1.073 g/L)分离获得的大鼠骨髓基质细胞并传代扩增,用二甲亚砜(DMSO),丁羟回醚(BHA)诱导分化,采用倒置相差显微镜观察细胞形态学变化,分化后细胞用神经丝蛋白(NF)抗体,胶质源性纤维酸性蛋白(GFAP)抗体作细胞免疫荧光检测?采用半定量RT-PCR检测分化前和分化后2 h,6 h,12 h,24 h和48 h神经生长因子(NGF),脑源性神经因子(BDNF),神经营养因子-3(NT-3)及其相应受体TrkA,TrkB,TrkC和神经营养因子低亲和力受体p75NTR的表达变化?结果:①骨髓基质细胞体外诱导分化后2 h即出现形态学变化,表现为细胞胞体呈球形,突起增多,形态类似神经元?细胞免疫荧光检测显示神经丝蛋白(NF)抗体标记的神经元细胞阳性,GFAP阴性?②在骨髓基质细胞分化前后都有NGF和BDNF持续表达,但分化后NGF表达降低,TrkA,TrkB在分化前细胞中不表达,在诱导分化开始后12 h可以检测到他们的表达,而NT-3,TrkC在分化前后都不表达?③p75NTR在诱导分化后6 h开始出现表达,12 h后显著降低并很快消失?结论:骨髓基质细胞在体外可以定向诱导为神经细胞,诱导分化前后神经营养因子(NT)及其Trk受体和p75受体的表达发生了显著变化,NT与Trk受体及p75受体作用的信号途径可能在骨髓基质细胞分化为神经细胞过程中起重要作用?
英文摘要:
      Objective:To study the gene expression pattern of neurotrophins and their receptors during neuronal differentiation of rat bone marrow stromal cells(BMSCs) in vitro and explore the mechanism of differentiation from bone marrow stromal cells into neurons. Methods:BMSCs were separated from rat bone marrow using percoll(1.073 g/L) reagent and cultured in vitro. The induction of BMSCs differentiating into neurons was used by dimethylsulfoxide(DMSO) and butylated hydroxyanisole(BHA), and the morphological changes were observed and recorded under inverted microscope. The immunofluorescence method was used to identify the differentiated BMSCs which would express neuronal marker. The expression levels of neurotrophines NGF,BDNF ,NT-3 and their high or low-affinity(TrkA,TrkB,TrkC,p75NTR) receptors before and at 2,6,12,24 and 48 h after differentiation of BMSCs were investigated by RT-PCR. Results:①Two hours after the induction,the differentiated BMSCs had morphological changes into ball-like shape with short prominence,like neurons. Immunofluorescence detection showed that the differentiated BMSCs expressed neuron marker neurofilament protein,but it was failed to detect the expression of astrocyte marker glial fibrillary acidic protein under this induction condition.②The NGF and BDNF mRNA could be detected in BMSCs during the whole stage,and the expression levels of NGF mRNA gradually decreased after induction. RT-PCR also revealed that both TrkA and TrkB expression were detected at 12 h following the onset of differentiation while not detected before differentia-tion. However no detectable expression of NT-3 and TrkC was observed in those cells either before or during differentiation. ③p75NTR appeared about 6 h after the induction of differentiation and persisted up to 12 h time point,but significantly declined and quickly diminished thereafter. Conclusion:BMSCs can be induced to differentiate into neurons in vitro. Significant changes of the expressions of neurotrophins(NT) NGF,BDNF,NT-3 and their high or low-affinity(TrkA,TrkB,TrkC,p75NTR) receptors were observed when BMSCs undergoing neuronal differentiation. The signaling pathway of NT and their receptors may take an important role in the differentiation of BMSCs into neurons.
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