摘要
目的:检测由骨髓源性神经干细胞诱导分化而成的神经元样细胞,能否分泌γ-氨基酸(γ-aminobutyricacid,GABA)神经生物活性物质成分,从而初步推断是否具有神经元的生化特征。方法:无菌条件下抽取新西兰大白兔骨髓,梯度密度离心分离获取骨髓基质细胞(bonemarrowstromacells,BMSCs),在神经干细胞培养基及脑源性神经营养因子(BDNF)等条件下,于体外诱导分化为神经干细胞及神经元样细胞,采用免疫细胞化学方法进行鉴定,并应用高效液相色谱法(HPLC)检测GABA生物活性物质的含量。结果:BMSCs培养12d可见细胞大而圆,免疫细胞化学检测巢蛋白抗原阳性;培养20d可见具有长突起的神经元样细胞形成,神经元核蛋白免疫细胞化学检查阳性,HPLC检测骨髓源性神经元样细胞含有高浓度GABA神经递质成分。方差分析显示:随着BMSCs培养天数的增加,其所含的GABA浓度也渐增加,由每1×107细胞中(3.43±0.25)μmol/L增至(14.69±3.51)μmol/L(F=23.69,P=0.0014)。结论:兔BMSCs在体外条件下可以增殖分化,前期表达Nestin,分化的细胞则可表达NeuN特异性抗体,并含有高浓度的类GABA神经递质成分。
AIM:To study whether the neuron like cells derived from bone marrow stromal cells(BMSCs)in vitro,can excrete neural biological activity material,γ aminobutyric acid(GABA),and then to deduce whether they possess the biochemistry characters of neuron. METHODS:Under the sterile condition,BMSCs,which were isolated from bone marrow of New Zealand rabbits by gradient centrifugalization,were induced and differentiated into neural stem cells and neuron like cells by nerve stem cell culture medium and brain derived neurotrophic factor(BDNF) in vitro.All of the differentiated cells were evaluated by immunocytochemical method,and high performance liquid chromatograpy(HPLC) technology was used to measure the content of GABA neural biological activity materials. RESULTS:BMSCs showed magnus and round shape 12 days after culture and expressed 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,there was a high content of GABA neurotransmitters in neuron like cells derived from BMSCs.Analysis of variance showed that the content of GABA gradually increased from(3.43±0.25) to(14.69±3.51) μmol/L per 1×107 cells following the prolongation of culture time(F=23.69,P=0.001 4). CONCLUSION:BMSCs of rabbit can be proliferated and differentiated in vitro and express Nestin positive antigen in the prophase,and the differentiated cells can express NeuN positive antigen in the anaphase,containing the high concentration of GABA,a kind of neurotransmitter.
出处
《中国临床康复》
CSCD
2004年第10期1854-1855,T001,共3页
Chinese Journal of Clinical Rehabilitation
基金
国家自然科学基金(30270491)
全军医学科研"十五"计划重大课题(01Z054)
广东省自然科学基金项目犤粤基办(2000)25犦
广东省科技计划项目犤粤企2001(367)犦~~
