淫羊藿素诱导小鼠胚胎干细胞体外定向分化为神经细胞

目的:采用淫羊藿素(icaritin,ICT)改变小鼠胚胎干细胞(embryonic stem cell,ES细胞)体外培养微环境,论证ICT提高ES细胞体外定向分化为神经细胞的效应。方法:采用拟胚体培养法,评价ICT对ES细胞体外定向分化为神经细胞的诱导作用;并利用RT-PCR法和免疫荧光法鉴定神经细胞特异基因和蛋白表达谱。结果:ICT在10-7mol/L浓度时,对ES细胞定向分化为神经细胞表型呈现最佳诱导效应,在分化d8 8时,分化率高达80%(P<0.001),并呈良好的量效和时效关系。分化神经表型者表达神经元特异性微管蛋白(β-tubulin Ⅲ)基因和神经胶质细胞特异性胶质纤维酸性蛋白(GFAP)基因,同时伴有神经前体细胞特异性标志蛋白(nestin)及β-tubulin Ⅲ和GFAP特异性蛋白阳性表达。结论:应用拟胚体培养微环境调控法,ICT可诱导小鼠ES细胞定向分化为神经细胞,并与神经发育依赖性特异基因和蛋白表达呈正相关。

Directed differentiation of mouse embryonic stem cells into neuronal cells induced by icaritin in vitro

OBJECTIVE: To evaluate the inductive effects of icaritin (ICT) on the directed differentiation of mouse embryonic stem (ES) cells into neuronal cells in vitro. METHODS: ES cells were cultured with embryoid body (EB) formation cultures, ICT in different concentrations was added in the cultural media and the cells were harvested in several differentiation phases. The expression spectrums of neuronal cell-specific genes and proteins were verified by semi-quantitative RT-PCR and immunocytochemistry analysis, respectively. RESULTS: Differentiation of neurocyte phenotype from ES cells was promoted by ICT in a concentration-and time-dependent manner. ICT at 10(-7)mol/L significantly enhanced the differentiation toward neuronal cells, and up to 80 % of EBs outgrowth in d 8+8 incubation. The gene expressions of beta-tubulin III in neuron and GFAP in glial cells were detected in neuronal cell phenotype derived from EBs. Furthermore, nestin was detected in precursor cells, beta-tubulin III and GFAP were detected in the generated precursor neurocytes immunocytochemically. CONCLUSION: Directed differentiation of neurons is facilitated by ICT in EB formation culture, which is associated with the expression of developmental-dependent gene and protein.