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目的:通过胚胎中脑祖细胞(MPC)体外培养获得高纯度的多巴胺神经元培养体系。方法:取自E12鼠胚中脑腹侧的MPC悬液在添加bFGF的DMEM/F12/N2/L-抗坏血酸-2-磷酸酯倍半镁盐(AA-2P)培养液中扩增培养,5d后停用bFGF,促进细胞分化,4d后通过Neurobasal/阿糖胞苷(Ara-c)抑制胶质细胞生长,获得纯神经元,使用TH鉴定细胞,计数细胞比例和纯度,β-tubulinIII鉴定成熟神经元,并计数细胞比例和纯度。结果:在添加了bFGF20μg/L的DMEM/F12/N2/AA-2P培养液中MPC增殖良好,体外培养7d后细胞数量扩增到培养前的(16·54±1·25)倍;使用Neu-robasal/阿糖胞苷后胶质细胞受抑制,神经元占94%以上,其中多巴胺(DA)能神经元的比例约(35·56±4·13)%。结论:E12鼠胚MPC原代培养合并使用阿糖胞苷是获取高纯度DA能神经元培养体系的可靠途径。
OBJECTIVE: To obtain a high purity dopamine neuron culture system by culturing embryonic mesogeny progenitor cells (MPC) in vitro. Methods: The MPC suspension from the ventral midbrain of E12 mouse embryos was cultured in DMEM / F12 / N2 / L-ascorbic acid-2-phosphate sesqui-methylate (AA-2P) medium supplemented with bFGF, After 4 hours, neurons were inhibited by Neurobasal / Cytarabine (Ara-c) to obtain pure neurons. TH cells were counted, the proportion and purity of cells were counted, β-tubulinIII was used to identify the mature Neurons, and count the cell ratio and purity. Results: MPC proliferated well in DMEM / F12 / N2 / AA-2P supplemented with 20μg / L bFGF, and the number of cells proliferated after cultured in vitro for 7 days (16.54 ± 1.25 times) -robasal / cytarabine, the percentage of neurons in dopaminergic neurons was about 35.56 ± 4.13%. CONCLUSION: Cytosine arabinoside (MPC) primary culture of E12 murine embryos combined with cytarabine is a reliable method for obtaining high purity DA neuron culture system.