本文選題：神經(jīng)干細(xì)胞　切入點(diǎn)：定向分化　出處：《陜西師范大學(xué)》2009年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】： 目的:在探討神經(jīng)干細(xì)胞(neural stem cells,NSCs)分離和培養(yǎng)的基礎(chǔ)上,研究中藥黃芪(Radix Astragali)對(duì)體外培養(yǎng)的NSCs分化方向的影響,檢測(cè)在誘導(dǎo)過(guò)程中相關(guān)基因nestin、neuroD、ngn2和Mash-1的動(dòng)態(tài)表達(dá)變化,初步探索神經(jīng)干細(xì)胞的分化機(jī)制,為中藥誘導(dǎo)神經(jīng)干細(xì)胞體外定向分化的研究提供初步的實(shí)驗(yàn)依據(jù)。 方法:(1)從懷孕15天的小鼠胎腦組織中分離NSCs,采用無(wú)血清培養(yǎng)法進(jìn)行NSCs的體外培養(yǎng)。每天倒置相差顯微鏡下觀察細(xì)胞形態(tài),通過(guò)繪制細(xì)胞生長(zhǎng)曲線,觀察并驗(yàn)證所培養(yǎng)的細(xì)胞具有自我更新和增殖能力的干細(xì)胞特性。采用免疫細(xì)胞化學(xué)法檢測(cè)NSCs標(biāo)志蛋白-神經(jīng)上皮干細(xì)胞蛋白(neural epithelial stem protein,Nestin)的表達(dá)。(2)采用無(wú)血清培養(yǎng)技術(shù)得到的第2代神經(jīng)干細(xì)胞球作為實(shí)驗(yàn)細(xì)胞,在培養(yǎng)液中分別加入不同濃度的黃芪注射液,2mg/ml、20mg/ml、100mg/ml、200mg/ml和空白對(duì)照組。每天觀察不同濃度黃芪對(duì)神經(jīng)球的影響,確定影響最大的濃度作為下一步的實(shí)驗(yàn)濃度。在傳代2次后的神經(jīng)干細(xì)胞內(nèi)加入該濃度黃芪后,進(jìn)行免疫細(xì)胞化學(xué)檢測(cè)神經(jīng)元特異性烯醇化酶(neuron specific enolase,NSE)和膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein,GFAP)的表達(dá),計(jì)算黃芪組和對(duì)照組誘導(dǎo)分化為NSE陽(yáng)性細(xì)胞的比率。(3)選取加黃芪后1天、3天、5天做為觀察和研究的時(shí)間段,采用RT-PCR的方法分析神經(jīng)干細(xì)胞分化后各個(gè)時(shí)期Nestin及內(nèi)源性bHLH轉(zhuǎn)錄因子家族基因neuroD、ngn2和Mash-1在分化過(guò)程中表達(dá)的差異。 結(jié)果:(1)我們從胚胎小鼠腦組織中分離的細(xì)胞在無(wú)血清的培養(yǎng)液中得到了懸浮的神經(jīng)球。神經(jīng)球具有自我更新和表達(dá)Nestin的能力。(2)與對(duì)照組比較,黃芪能增加NSCs向細(xì)胞元分化的比率(p＜0.05)。(3)RT-PCR結(jié)果顯示:在神經(jīng)干細(xì)胞分化的過(guò)程中,實(shí)驗(yàn)組Nestin在分化后5天的過(guò)程中持續(xù)表達(dá),bHLH基因neuroD、ngn2和Mash-1在第一天表達(dá)最強(qiáng),以后逐漸減弱。說(shuō)明:在黃芪誘導(dǎo)神經(jīng)干細(xì)胞分化的過(guò)程中,可能啟動(dòng)了bHLH轉(zhuǎn)錄因子家族,從而使神經(jīng)干細(xì)胞向神經(jīng)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.
【學(xué)位授予單位】：陜西師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類(lèi)號(hào)】：R329

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