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目的:建立体外培养和诱导嗅黏膜神经干细胞向酪氨酸羟化酶阳性神经元分化的方法,探讨该细胞的干性和分化特性。方法:首先用含10%胎牛血清的DMEM/F-12混合培养基培养成年大鼠嗅黏膜细胞,当细胞贴壁后将培养基更换为含表皮生长因子(epidermal growth factor,EGF)和碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF),不含血清的干细胞培养基,以促进干细胞增殖形成神经球。将神经球取出用干细胞培养基悬浮培养以纯化扩增神经干细胞。应用干细胞相关蛋白Nestin及CD133的抗体对神经干细胞进行免疫荧光染色,观察上述蛋白在细胞的表达。用Neurobasal培养基(含B27、NGF、ATRA及SHH)培养扩增后的神经干细胞,诱导其向神经元分化。用神经丝蛋白(neurofilament-200,NF-200)和酪氨酸羟化酶(tyrosine hydroxylase,TH)抗体对分化的细胞进行免疫荧光染色;用免疫印迹法检测上述细胞裂解样品内的相应标志蛋白的电泳条带。结果:嗅黏膜神经干细胞高表达Nestin及CD133两种神经干细胞标志蛋白;干细胞经诱导分化后其形态具有神经元特征,并且高表达NF和TH两种神经细胞标志蛋白。结论:贴壁/悬浮序贯培养方法可体外纯化扩增嗅黏膜神经干细胞,该细胞可分化为酪氨酸羟化酶阳性神经元。
OBJECTIVE: To establish a method for culturing and inducing neural stem cells of olfactory mucosa to differentiate into tyrosine hydroxylase positive neurons in vitro, and to investigate the characteristics of stem cell differentiation and differentiation. Methods: Adult rat olfactory mucosa cells were cultured in DMEM / F-12 medium supplemented with 10% fetal bovine serum. When the cells adhered to each other, the medium was changed to medium containing epidermal growth factor (EGF) and base Basic fibroblast growth factor (bFGF), serum-free stem cell culture medium to promote the proliferation of stem cells to form neurospheres. The neurospheres are removed and cultured in stem cell medium to purify the expanded neural stem cells. The neural stem cells were immunofluorescently stained with antibodies to Nestin and CD133 to observe the expression of these proteins in the cells. The cultured neural stem cells were cultured in Neurobasal medium (containing B27, NGF, ATRA and SHH) to induce neuronal differentiation. Differentiated cells were immunofluorescently stained with neurofilament-200 (NF-200) and tyrosine hydroxylase (TH) antibodies; Western blotting was used to detect the corresponding marker proteins in the cell lysis samples Of the electrophoresis band. Results: Olfactory mucosal neural stem cells expressed high levels of Nestin and CD133 neural stem cell marker proteins. After induced by stem cells, their morphological characteristics were neurons, and expressed high levels of NF and TH neuronal marker proteins. Conclusion: Adherent / suspension sequential culture method can purify and amplify olfactory mucosa neural stem cells in vitro, which can differentiate into tyrosine hydroxylase positive neurons.