本文選題：神經干細胞　切入點：定向分化　出處：《陜西師范大學》2009年碩士論文　論文類型：學位論文

【摘要】： 目的:在探討神經干細胞(neural stem cells,NSCs)分離和培養(yǎng)的基礎上,研究中藥黃芪(Radix Astragali)對體外培養(yǎng)的NSCs分化方向的影響,檢測在誘導過程中相關基因nestin、neuroD、ngn2和Mash-1的動態(tài)表達變化,初步探索神經干細胞的分化機制,為中藥誘導神經干細胞體外定向分化的研究提供初步的實驗依據。 方法:(1)從懷孕15天的小鼠胎腦組織中分離NSCs,采用無血清培養(yǎng)法進行NSCs的體外培養(yǎng)。每天倒置相差顯微鏡下觀察細胞形態(tài),通過繪制細胞生長曲線,觀察并驗證所培養(yǎng)的細胞具有自我更新和增殖能力的干細胞特性。采用免疫細胞化學法檢測NSCs標志蛋白-神經上皮干細胞蛋白(neural epithelial stem protein,Nestin)的表達。(2)采用無血清培養(yǎng)技術得到的第2代神經干細胞球作為實驗細胞,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和空白對照組。每天觀察不同濃度黃芪對神經球的影響,確定影響最大的濃度作為下一步的實驗濃度。在傳代2次后的神經干細胞內加入該濃度黃芪后,進行免疫細胞化學檢測神經元特異性烯醇化酶(neuron specific enolase,NSE)和膠質纖維酸性蛋白(glial fibrillary acidic protein,GFAP)的表達,計算黃芪組和對照組誘導分化為NSE陽性細胞的比率。(3)選取加黃芪后1天、3天、5天做為觀察和研究的時間段,采用RT-PCR的方法分析神經干細胞分化后各個時期Nestin及內源性bHLH轉錄因子家族基因neuroD、ngn2和Mash-1在分化過程中表達的差異。 結果:(1)我們從胚胎小鼠腦組織中分離的細胞在無血清的培養(yǎng)液中得到了懸浮的神經球。神經球具有自我更新和表達Nestin的能力。(2)與對照組比較,黃芪能增加NSCs向細胞元分化的比率(p＜0.05)。(3)RT-PCR結果顯示:在神經干細胞分化的過程中,實驗組Nestin在分化后5天的過程中持續(xù)表達,bHLH基因neuroD、ngn2和Mash-1在第一天表達最強,以后逐漸減弱。說明:在黃芪誘導神經干細胞分化的過程中,可能啟動了bHLH轉錄因子家族,從而使神經干細胞向神經元方向分化。
[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.
【學位授予單位】：陜西師范大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R329

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