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目的探讨诱导脂肪源性神经干细胞(neural stem cells,NSCs)分化为GABA能神经元的体外培养方法,为细胞移植修复神经系统损伤提供种子细胞。方法贴壁培养C57BL/6小鼠(附睾脂肪垫)脂肪来源的干细胞(adipose-derived stem cells,ADSCs),流式细胞仪进行表型鉴定(CD45、CD90、CD105)及标志物纤连蛋白(fibronectin)鉴定后,在碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)、表皮生长因子(epidermal growth factor,EGF)、2%B27的Neuro-basal培养基中诱导成NSCs。用免疫荧光细胞染色法进行NSC特异性标记物Nestin、增殖能力BrdU检测,再次添加脑源性神经营养因子、骨形成蛋白2后,维甲酸(RA)诱导分化为γ-氨基丁酸能神经元,并进行其标志物GABA的检测。结果分离纯化的ADSCs CD90、CD105表达强阳性,CD45表达阴性;诱导后的干细胞球经Nestin及BrdU鉴定为NSCs,且具有较强的增殖能力;神经干细胞诱导分化后的神经元GABA抗体染色呈阳性。结论在特定神经营养因子作用下,ADSCs在体外诱导生成的NSCs可被诱导分化为GABA能神经元。
Objective To investigate the in vitro culture method of differentiating adipose derived neural stem cells (NSCs) into GABAergic neurons and to provide seed cells for cell transplantation to repair nervous system injury. Methods Adipose-derived stem cells (ADSCs) of C57BL / 6 mice (epididymal fat pad) were adherently cultured. The phenotypes were identified by flow cytometry (CD45, CD90, CD105) and the marker fibronectin fibronectin), NSCs were induced in basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and 2% B27 in Neuro-basal medium. NSC specific marker Nestin was detected by immunofluorescence staining, BrdU assay was used to detect the proliferative capacity. After adding BDNF and BMP 2 again, retinoic acid (RA) was induced to differentiate into GABAergic neurons , And the detection of its marker GABA. Results The CD90 and CD105 expression of ADSCs was strongly positive and CD45 was negative. The induced stem cell spheres were identified as NSCs by Nestin and BrdU, and had strong proliferative ability. Neuronal GABA-positive cells stained positive . Conclusion Under the action of specific neurotrophic factor, NSCs induced by ADSCs in vitro can be induced to differentiate into GABAergic neurons.