星形胶质细胞条件培养液促进胚胎干细胞分化为神经元样细胞的实验研究

目的:探讨星形胶质细胞条件培养液在体外能否促进胚胎干细胞（ESCs）向神经元方向分化。 方法:在Bain等建立的4-/4+方案基础上，分成两组：单用ATRA组和ATRA联合胎脑星形胶质细胞条件培养液组，分3阶段诱导ESCs定向生成神经元样细胞。形态学观察及畸胎瘤形成试验对ESCs的全能性进行鉴定；免疫组化染色法检测nestin、GFAP、NSE和NF-200的表达对诱导过程进行动态监测。 结果:（1）体外培养的小鼠ESC-D3细胞在胚胎成纤维饲养层细胞上连续传代培养，仍保持向3胚层分化的能力。（2）在诱导分化阶段，联合诱导组分化细胞NF-200和NSE的表达阳性率高于单用ATRA组。（3） 联合诱导组最终诱导ESCs生成纯度高达73.5%神经元样细胞。 结论:采用胚胎脑星形胶质细胞条件培养液可较高产率及纯度地促进ESCs生成神经元样细胞，值得推广应用。

Efficient production of neuron-like cells from embryonic stem cells induced by astrocyte-conditioned medium in vitro

AIM: To investigate whether efficient production of neuron-like cells from embryonic stem cells(ESCs) can be indued by astrocyte-conditioned medium(ACM) in vitro.METHODS: Based on the 4-/4 protocol established by Bain,two groups were studied: ATRA group,ATRA with ACM group.ESCs were induced into neuron-like cells by means of three-step differentiation in vitro.The totipotency of ESCs was identified by the observation of cells' morphology and the formations of teratoma in immunocomprised mice.The cell differentiation was evaluated continuously by detection of the cellular specific markers of neural stem cell, neurons and astrocytes such as nestin,NF-200,NSE and GFAP using immuno-histochemistry assay.RESULTS:(1) The ESC-D3 cells kept the ability of differentiation into cellular derivations of all three primary germ layers after continuous passage culture.(2) The ratio of NF-200 and NSE positive cells in the cells induced by ATRA with ACM was higher than that in the cells induced by ATRA only.(3) Finally,the positive rate of the neuron-like cells was up to 73.5% in the group induced by ATRA with ACM.CONCLUSION: The ESCs are induced into neuron-like cells with high purity and efficiency by ATRA with ACM.