摘要
目的研究体外利用全反视黄酸(RA)诱导小鼠胚胎干细胞(mESC)分化为神经细胞的培养方法。方法悬滴3天转悬浮1天两者结合的培养方法得到胚体(EBs),再利用RA对其处理4天后,进行免疫组织化学、RT-PCR以及兴奋性功能的检测,观察神经细胞获得的效率及其生理特性。结果利用RA诱导方法得到的神经细胞,免疫组化表明nestin蛋白表达的阳性率为(53.49±6.02)%,且RT-PCR法分析其能表达nestin、glutaminase和Brn-3等神经细胞特异基因,同时在Glu刺激下具有与脑来源神经元相似的特性和功能。结论利用本实验诱导方法,获得神经细胞的比例较高,且具有与神经元相似的生物学特性。
Objective To investigate the probabilities and properties of embryonic stem（ES） cells differentiating into neuronal cells in vitro by retinoic acid.Methods Mouse ES D3 cells were cultured by means of handing drop and suspending on the ES conditioned media for 3 days and 1day until ES cell aggregations was formed（embryonic bodies,EBs）.The EBs were exposed to retinoic acid（RA） for another 4 days.Immunohistochemistry,RT-PCR assay and the fluorescence intensity changes of calcium after Glu stimulation were performed to identify the characteristics and differentiation efficiency of the differentiated cells.Results Expression of nestin protein was detected by immunohistochemistry after 96 h induction and the nestin positive cells were（53.49±6.02） %.RT-PCR assay further identified that the induced cells expressed transcripts for neural associated genes including nestin、 glutaminase and Brn-3.And the induced cells have the same character with neuronals after Glu stimulation.Conclusion ES cells can be successfully induced by retinoic acid into neuronal cells expressing multiple properties of neurons in vitro,and these cells have the similar characters with the neuronal derived in vivo.
出处
《卫生研究》
CAS
CSCD
北大核心
2010年第5期545-549,共5页
Journal of Hygiene Research
基金
"十一五"国家科技支撑计划重大项目(No2006BAK02A07)
农业部科技行业科技项目(农药风险评估综合配套技术研究)(No200903054-06)
中国疾病预防控制中心青年科研基金资助项目(No2010A206)
关键词
胚胎干细胞
视黄酸
神经分化
embryonic stem cells
retinoic acid
neuronal differentiation
characteristic
