间充质干细胞向神经细胞诱导过程中表面标记的变化(英文)

Surface marker changes during induced differentiation of bone marrow mesenchymal stem cells into neuronal-like cells

背景:成熟的中枢神经系统缺乏再生能力。因此,找寻新的神经干细胞来源就显得尤为重要。目的:观察碱性成纤维细胞生长因子和表皮生长因子对间充质干细胞诱导分化为神经元样细胞的差异。方法:从正常人骨髓中分离获取间充质干细胞,体外培养扩增、纯化后种植于96孔板中,调整细胞密度为0.25×108L-1,每孔200μ LDMEM/F12培养液,从第0～7天,每天于固定时间在5个孔里加入20μ LMTT,放入培养箱继续培养4h后吸净孔内的培养液,加入二甲基亚砜100μL/孔。另外,一部分细胞用碱性成纤维细胞生长因子和表皮生长因子单独或联合对其进行诱导分化。MTT法绘制细胞的生长曲线,RT-PCR比较两种细胞因子诱导细胞的差异,TRAP(PCR)-ELISA检测诱导后细胞的端粒酶活性。结果与结论:未诱导的第4代间充质干细胞有nestin,GFAP和NF-M mRNA的表达,随着诱导时间延长表达逐渐加强,但是诱导7d后nestin的表达量逐渐下降。MAP2 mRNA的表达在诱导后出现,随着诱导,表达逐渐加强。碱性成纤维细胞生长因子+表皮生长因子诱导组和碱性成纤维细胞生长因子诱导组MAP2 mRNA的表达量明显多于表皮生长因子诱导组,而表皮生长因子组可见较多胶质纤维酸性蛋白的表达。提示间充质干细胞可以在体外培养,扩增以及向神经干细胞分化。分化后的神经干细胞具有增殖的活性,但是不具备肿瘤细胞的无限增殖性。

BACKGROUND：Adult central nervous system lacks the ability to regenerate,so it is of great significance to find a new source of neural stem cells.OBJECTIVE：To investigate the differences between basic fibroblast growth factor（bFGF） and epidermal growth factor（EGF） in inducing bone marrow mesenchymal stem cells（MSCs） to differentiate into neurons in vitro.METHODS：MSCs were isolated from normal human bone marrow using density gradient centrifugation and cell attachment method.MSCs were plated in 96-well culture plates at a density of 0.25×108/L and cultured with 200 μL DMEM/F12 for 0,1,2,3,4,5,6,and 7 days,with 20 μL MTT（5 mg/mL） in 5 wells at each time point.The supernatant was removed and 100 μL dimethyl sulphoxide was added to each well for 4 additional hours of incubation.In addition,some cells were exposed to bFGF and EGF.Growth curve was determined with MTT method.Telomerase activity were examined by TRAP（PCR）-ELISA.Additionally,the functional differences of the two cytokines were checked by RT-PCR.RESULTS AND CONCLUSION：RT-PCR revealed that nestin,glial fibrillary acidic protein（GFAP） and neurofilament subunit M（NF-M） mRNA were expressed in un-induced MSCs of passage 4.Nestin expression reduced at 7 days.The expression of micro-tubule-associated protein-2（MAP2） mRNA was not detected until the induction,and increased thereafter.The expression of MAP2 mRNA was greater in bFGF＋EGF and bFGF alone groups compared with EGF alone group,and the expression of GFAP in EGF alone group was greater than other groups.Results showed that MSCs can be cultivated,proliferated and differentiated into neural stem cells in vitro.The differentiated neural stem cells have the activity of proliferation,but not have the ability of infinite proliferation as tumor cells.