兔骨髓源性神经干细胞分化为成熟神经元样细胞的特征研究

Characteristic study on the differentiation of rabbit bone marrow stromal cells into neuron-like cells

目的:探讨兔骨髓源性神经干细胞(bonemarrowstromalcells,BMSCs)经体外培养及诱导分化成的神经元样细胞,能否合成分泌5-羟色胺神经生物活性物质成分。方法:分离兔BMSCs,应用神经干细胞培养液和分化诱导因子进行体外培养和诱导分化,免疫细胞化学鉴定;并应用高效液相色谱法(HPLC)检测其5-羟色胺生物活性物质的含量。细胞培养所涉及的细胞因子主要有脑源性神经营养因子(BDNF)和维A酸。结果:BMSCs培养12d可见大而圆细胞,免疫细胞化学鉴定Nestin抗原阳性;培养20d可见长突起神经元样细胞,神经核蛋白(NeuN)免疫细胞化学检查阳性,高效液相检测BMSCs、空白神经干细胞培养液及L-02肝细胞阴性对照组未测到5-羟色胺生物活性物质,经体外培养增殖14d的神经干细胞及培养液、体外诱导分化20d的神经元样细胞及培养液则可检测到5-羟色胺分别为:每1×107细胞中(1.7940±0.0095),(4.010±0.1170)μg/L,差异有非常显著性意义(t=30.6323,P=0.001)。方差分析显示:随着骨髓基质细胞培养天数的增加,其所含的5-羟色胺浓度也渐增加,组间5-羟色胺含量差异有显著意义(P<0.01)。结论:兔BMSCs能在体外增殖,并表达Nestin抗原;而且,在适宜条件下能分化成神经元样细胞,并表达成熟神经元特异性核蛋白,合成、分泌5-羟色胺神经递质。

AIM:To study whether the neuron-like cells derived from bone marrow stromal c ells(BMSCs)in vitro,can excrete neural biological activity material,5-hydrox ytryptamine(5-HT),or not. METHODS:Under the sterile condition,BMSCs,which were isolated from bone marrow of New Zealand rabbits, were cultured in culture medium of neural stem cell(NSC ),induced and differentiated into neuronal-like cells with induced and differen tiated factors in vitro.All of the differentiated cells were evaluated by immuno cytochemical method,and high performance liquid chromatograpy(HPLC) technology w as used to measure the content of 5-HT neural biological activity materials.The cytokines during culture mainly included brain-derived neurotrophic factor(BDN F) and retinoic acid(RA). RESULTS:BMSCs showed magnus and round shape at 12 days after culture and expre ssed Nestin-positive antigen,some of which might be differentiated into neuron -like cells with long process and show neural nuclear protein(NeuN) -positive antigen at 20 days.Meanwhile,HPLC test showed that there were no 5-HT neural bi ological activity materials in BMSCs,free culture medium of NSC and L-02 hepato cyte(negative control);but 5-HT was found in NSC and its medium 15 days after c ulture in vitro and in neuron-like cells and its medium 20 days after different iation in vitro, which was(1.794 0±0.009 5) and(4.010±0.117 0) per 1×107 cell s,respectively.Analysis of variance showed that the content of 5-HT gradually i ncreased with the prolongation of culture time,and differences in the content of 5-HT were significant among the groups(P< 0.01). CONCLUSION:BMSCs of rabbit can proliferate in vitro and express Nestin-positi ve antigen,and moreover they can differentiate into neuronal-like cells under a appropriate condition,and the differentiated cells can express NeuN-positive a ntigen in the mature stage,synthesize and excrete 5-HT neurotransmitters.