体外诱导儿童骨髓间质干细胞分化为神经细胞

Generation of neural cells from children bone marrow stromal cells in vitro

目的 探讨儿童骨髓间质干细胞诱导分化为神经干细胞及神经细胞的方法及生物学特征 ,为将来进一步研究及利用其实验治疗小儿脊髓栓系综合征及其他神经系统疾病动物模型奠定基础。方法 取健康儿童骨髓离心初步分离骨髓间质干细胞 ,在α MEM培养基中进行无分化培养 12代进一步纯化扩增 ,加入预诱导液、诱导液进行定向诱导分化 ,逐日观察细胞形态变化 ,采用免疫组织化学技术对诱导后的细胞进行神经干细胞特异性抗原nestin、神经丝蛋白 β tublinШ和胶质纤维酸性蛋白 (GFAP)表达的定性检测 ,观察其表达情况 ;并采用RT PCR技术对不同时段神经干细胞特异性抗原nestin和神经生长因子受体TrkB (neurotrophicreceptortyrosinekinaseB ,TrkB)mRNA的表达进行检测 ,用上述两种技术手段 ,从不同角度和层面验证和初步探索BSMCs定向诱导分化为神经组织样细胞的细胞生物学和分子生物学特征。结果 骨髓间质干细胞经过 7d体外诱导 ,大约有 35 %的细胞发生形态学变化。免疫组化结果显示 ,细胞形态逐步发生变化并最终形成神经样的细胞 ,在诱导后30min表达神经干细胞特异性标志nestin抗原、7d时分别表达神经元特异性标志 β tublinⅢ抗原和星形胶质细胞特异性标志GFAP抗原。RT PCR检测结果也表明 ,被诱导后 5 .5h在mRNA水?

Objective To investigate the feasibility of generation of neural stem cells （NSCs） and neural cells from children bone marrow stromal stem cells （BMSCs） in vitro. Methods The BMSCs, voluntarily donated by a fourteen years old girl, were originally cultured in α-MEM supplemented with 20% FBS, 2mM L-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, and 25 ng/ml amphotericin B. After 12 passages, cells were cultured in the inductive media which were composed of DMEM, 2% dimethyl sulphoxide（DMSO） and 150μ MBHA. The expressions of specific antigens of NSCs including nestin, beta-tublin Ⅲ and glial fibrillary acidic protein in these cells were measured through immunohistochemical staining. Meanwhile, the mRNA levels of nectin and neurotrophic receptor tyrosine kinase （Trk B） were detected by RT-PCR. Results After induction by BHA for 7 days, networks of neurofilament were formed in about 35% of cells. The expression of beta-tublin Ⅲ and glial fibrillary acidic protein could be demonstrated in induced cells. Although the expression of nestin was transiently noted in these cells after 30 min of induction, that protein was undetectable after 6 days of culture. The mRNA of nectin was up-regulated after 5.5 hours of induction while it disappeared after 7 days of induction. The mRNA of trkB, however, was up-regulated both at 5. 5 hours and 6 days after induction. Conclusions It is feasibility to generate BMSCs and neuronal cells from children BMSCs.