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背景: 目前神经干细胞多由动物获得,不适合人类临床移植治疗。目的: 探索体外环境下人胚胎纹状体来源神经干细胞的培养方法,同时观察其生物学特性。方法: 取经水囊引产的孕8-16周人胚胎纹状体,体外用无血清DMEM培养基进行培养,待细胞形成神经球后进行传代,并应用含体积分数10%胎牛血清的DMEM/F12培养液进行诱导分化。结果与结论: 体外培养的人胚胎纹状体来源神经干细胞生长迅速,表达神经干细胞标志物nestin。克隆形成实验显示细胞克隆形成率为6.0%-7.0%;BrdU掺入实验显示细胞增殖率为37.9%。免疫荧光染色显示经诱导分化的细胞表达神经元标志物Ⅲ型β微管蛋白、星形胶质细胞标志物胶质纤维酸性蛋白及神经干细胞标志物nestin,但不表达少突胶质细胞标志物髓鞘碱性蛋白。可见人胚胎纹状体来源神经干细胞在体外无血清条件下可保持其生物学特点,具有自我更新能力,经胎牛血清诱导后可向神经元及星形胶质细胞分化。
Background: Currently, neural stem cells are mostly obtained from animals and are not suitable for human clinical transplantation. Objective: To explore the culture method of human embryonic striatum-derived neural stem cells in vitro and to observe its biological characteristics. Methods: Embryonic striatum of 8-16 weeks old pregnant women induced by the water sac was cultured in serum-free DMEM medium in vitro. After the neurospheres were formed, the cells were passaged. The cells were cultured in DMEM / F12 culture medium for differentiation. RESULTS AND CONCLUSION: Human embryonic striatum-derived neural stem cells cultured in vitro grew rapidly and expressed neural stem cell marker nestin. Clone formation experiments showed that the cell clone formation rate was 6.0% -7.0%; BrdU incorporation assay showed the cell proliferation rate was 37.9%. Immunofluorescence staining showed that the differentiated cells expressed neuronal markers type III beta tubulin, astrocyte marker glial fibrillary acidic protein and neural stem cell marker nestin, but did not express oligodendrocyte markers Myelin basic protein. It can be seen that human embryonic striatum-derived neural stem cells can maintain their biological characteristics under the condition of serum-free in vitro and have self-renewal ability. After being induced by fetal calf serum, neural stem cells can differentiate into neurons and astrocytes.