摘要
目的:在探讨神经干细胞(neural stem cells,NSCs)分离和培养的基础上,研究中药黄芪(Radix Astragali)对体外培养的NSCs分化方向的影响,检测在诱导过程中相关基因nestin、neuroD、ngn2和Mash-1的动态表达变化,初步探索神经干细胞的分化机制,为中药诱导神经干细胞体外定向分化的研究提供初步的实验依据。
方法:(1)从怀孕15天的小鼠胎脑组织中分离NSCs,采用无血清培养法进行NSCs的体外培养。每天倒置相差显微镜下观察细胞形态,通过绘制细胞生长曲线,观察并验证所培养的细胞具有自我更新和增殖能力的干细胞特性。采用免疫细胞化学法检测NSCs标志蛋白-神经上皮干细胞蛋白(neural epithelial stem protein,Nestin)的表达。(2)采用无血清培养技术得到的第2代神经干细胞球作为实验细胞,在培养液中分别加入不同浓度的黄芪注射液,2mg/ml、20mg/ml、100mg/ml、200mg/ml和空白对照组。每天观察不同浓度黄芪对神经球的影响,确定影响最大的浓度作为下一步的实验浓度。在传代2次后的神经干细胞内加入该浓度黄芪后,进行免疫细胞化学检测神经元特异性烯醇化酶(neuron specific enolase,NSE)和胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)的表达,计算黄芪组和对照组诱导分化为NSE阳性细胞的比率。(3)选取加黄芪后1天、3天、5天做为观察和研究的时间段,采用RT-PCR的方法分析神经干细胞分化后各个时期Nestin及内源性bHLH转录因子家族基因neuroD、ngn2和Mash-1在分化过程中表达的差异。
结果:(1)我们从胚胎小鼠脑组织中分离的细胞在无血清的培养液中得到了悬浮的神经球。神经球具有自我更新和表达Nestin的能力。(2)与对照组比较,黄芪能增加NSCs向细胞元分化的比率(p＜0.05)。(3)RT-PCR结果显示:在神经干细胞分化的过程中,实验组Nestin在分化后5天的过程中持续表达,bHLH基因neuroD、ngn2和Mash-1在第一天表达最强,以后逐渐减弱。说明:在黄芪诱导神经干细胞分化的过程中,可能启动了bHLH转录因子家族,从而使神经干细胞向神经元方向分化。
Objective:The aim of this study was to investigate the effects of the Radix Astragali on the differentiation of the neural stem cells(NSCs) through the neural stem cells isolation and culture, and to detect the dynamic expression change of the relational gene—nestin、neuroD、ngn2 and Mash-1 in the process of neuronal differentiation of the neural stem cell,and to explore some inner molecular mechanism of the differentiation.To provided the experimental bases for the induced differentiation of neurons in vitro.
Methods:(1) Cells were isolated from the 15d embryonic rat brain were cultured in the serum-free culture fluid.It was manifested that there were the morphological changes in the cells grown in the culture medium continuously can been seen under the inverted microscopy every day. Through the cell growth curve mapping to observe and verify that NSCs have the capacity of self-renewal and proliferation.Nestin is the markers of neural stem cell which were identified by chemoimmunity staining.(2) As the two generation neural stem cells were gained,they were separated into two groups:Radix Astragali were added into the experimental group,2mg/ml、20mg/ml、100mg/ml and 200mg/ml compared to the control group.To observe the morphological changes different concentrations of Radix Astragali on the effect of neurospheres and choice the best concentration of Radix Astragali for the next step.The cells we gained after added Radix Astragali for immunocytochemical detection of neuron-specific enolase(neuron specific enolase,NSE),glial fibrillary acidic protein(glial fibrillary acidic protein,GFAP) expression.Directly count the percentage of the neurons under the microscope.(3) The expression of the gene- nestin、neuroD、ngn2 and Mash-1 at the 1,3 and 5 days following the differentiation were detectd by RT-PCR.
Results:(1) We have gained the neurospheres from the 15d embryonic rat brain.NSCs have the ability of self-renew,and can be separated or purified in vitro under some proper conditions. The neural stem cell can express the Nestin marker protein.(2) Cell calculation showed that the amount of the neurons differentiated from the neural stem cells were more remarkable in the Radix Astragali group.(3) In the process of Neural stem cell differentiation,Nestin continuous expression in the experimental group after 5 days.The expression of the three bHLH gene- neuroD、ngn2 and Mash-1 in the experimental group following differentiation increased more obviously than in the control group compared.During the differentiation of the neural stem cells,the three bHLH genes were all up-regulated,especially in dyal.Later,gradually weakened.Our experiment shows that there does promoted the bHLH.
引文
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