黄芪在神经干细胞体外分化过程中作用的研究

发布时间：2018-03-12 10:52

本文选题：神经干细胞　切入点：定向分化　出处：《陕西师范大学》2009年硕士论文　论文类型：学位论文

【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。
[Abstract]:Aim: to investigate the effect of Radix Astragali on the differentiation of neural stem cells in vitro, and to detect the dynamic expression of the related genes Neisin neuroDngn2 and Mash-1 during the induction. To explore the differentiation mechanism of neural stem cells and to provide a preliminary experimental basis for the study of directional differentiation of neural stem cells induced by traditional Chinese medicine in vitro. Methods NSCs were isolated from fetal brain tissue of 15 days pregnant mice and cultured in vitro by serum-free culture. The morphology of NSCs was observed by inverted phase contrast microscope every day, and the cell growth curve was drawn. The characteristics of stem cells with self-renewal and proliferation were observed and verified. The expression of neural epithelial stem protein nestin, a NSCs marker protein, was detected by immunocytochemistry. The second generation of neural stem cell spheres obtained by the technique is used as experimental cells. Different concentrations of Astragalus membranaceus injection were added to the culture medium. The effects of different concentrations of Astragalus membranaceus injection on the neurospheres were observed every day by adding 20 mg / ml Astragalus membranaceus injection 100mg / ml or 200mg / ml to the control group. Determine the concentration that has the greatest effect as the next experimental concentration. After the second passage of neural stem cells, the concentration of Astragalus membranaceus is added to the neural stem cells. The expression of neuron specific enolase (specific) and glial fibrillary acidic protein (GFAPs) were detected by immunocytochemistry. The ratio of induced differentiation into NSE positive cells in astragalus group and control group was calculated. RT-PCR method was used to analyze the differences in the expression of Nestin and endogenous bHLH transcription factor gene neuroDngn2 and Mash-1 in the differentiation process of neural stem cells at different stages after differentiation. Results 1) the cells isolated from the brain of embryonic mice were cultured in serum-free medium. The neurospheres had the ability of self-renewal and expression of Nestin.) compared with the control group, the neurospheres had the ability of self-renewal and expression of Nestin. The results of RT-PCR showed that during the differentiation of neural stem cells, the expression of NSCs gene neuroDngn2 and Mash-1 was the strongest in the experimental group 5 days after differentiation. It is suggested that during the differentiation of neural stem cells induced by Astragalus membranaceus, the family of bHLH transcription factors may be initiated, thus the neural stem cells differentiate into neurons.
【学位授予单位】：陕西师范大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329

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