本文選題：藥物篩選 + 神經(jīng)干細(xì)胞�。� 參考：《吉林大學(xué)》2014年碩士論文

【摘要】：目前，神經(jīng)干細(xì)胞的定向誘導(dǎo)分化研究已經(jīng)成為生命科學(xué)領(lǐng)域中最引人注目的熱點(diǎn)。隨著干細(xì)胞定向誘導(dǎo)分化技術(shù)的發(fā)展，使得干細(xì)胞移植治療成為可能。那么，在體內(nèi)外研究定向分化的發(fā)生機(jī)制、尋找治療靶點(diǎn)、開發(fā)新的治療藥物，已成為現(xiàn)代醫(yī)學(xué)和生物學(xué)研究的重點(diǎn)問題。 乙酰膽堿廣泛存在于中樞神經(jīng)系統(tǒng)中，除此之外還存在于腸神經(jīng)與植物神經(jīng)中。乙酰膽堿(ACh)已被證明在中樞神經(jīng)系統(tǒng)（CNS）調(diào)節(jié)著不同的生理功能，包括認(rèn)知、注意力和覺醒等，在中樞神經(jīng)系統(tǒng)中膽堿能神經(jīng)元的機(jī)能失調(diào)也與年齡相關(guān)的記憶障礙、神經(jīng)系統(tǒng)退行性疾病阿爾茨海默�。ˋD）的發(fā)病相關(guān)。乙酰膽堿轉(zhuǎn)移酶（ChAT）催化乙酰輔酶A和膽堿生成乙酰膽堿，是膽堿能神經(jīng)元遞質(zhì)乙酰膽堿合成的限速酶。乙酰膽堿儲(chǔ)存于突觸前小泡，囊狀乙酰膽堿（VAChT）負(fù)責(zé)轉(zhuǎn)運(yùn)加載乙酰膽堿到這些囊泡。乙酰膽堿結(jié)合兩種不同類型的受體：煙堿乙酰膽堿受體（nAChR）和毒蕈堿型乙酰膽堿受體（mAChRs）。鑒于ChAT是神經(jīng)細(xì)胞重要的標(biāo)志物，我們以ChAT啟動(dòng)子為靶點(diǎn)建立篩選模型，對(duì)小分子化合物庫進(jìn)行篩選，以期發(fā)現(xiàn)能夠調(diào)節(jié)ChAT啟動(dòng)子活性的小分子化合物。 我們首先構(gòu)建了pGL3-ChAT啟動(dòng)子的熒光素酶報(bào)告載體，并將重組載體轉(zhuǎn)入P19和HEK293T細(xì)胞中檢測(cè)啟動(dòng)子活性，結(jié)果顯示，構(gòu)建的啟動(dòng)子具有較高的活性，，與對(duì)照相比在統(tǒng)計(jì)學(xué)上存在極顯著性差異（**P㩳0.01）。之后我們利用已建立好的熒光素酶報(bào)告基因法對(duì)實(shí)驗(yàn)室中的400多種小分子化合物進(jìn)行篩選，發(fā)現(xiàn)TI5可明顯激活ChAT啟動(dòng)子，與對(duì)照相比在統(tǒng)計(jì)學(xué)上存在極顯著性差異（*P㩳0.05），隨后，我們從mRNA水平和蛋白質(zhì)水平對(duì)化合物TI5的ChAT基因表達(dá)調(diào)控作用進(jìn)行了驗(yàn)證。為了闡明TI5促進(jìn)ChAT表達(dá)的分子機(jī)制，我們通過轉(zhuǎn)錄因子應(yīng)答性熒光素酶報(bào)告基因法對(duì)TI5的細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)途徑進(jìn)行了分析，發(fā)現(xiàn)化合物TI5能夠增加pSRE-TA-luc熒光素酶報(bào)告質(zhì)粒的活性，于是我們應(yīng)用免疫印跡法分析了TI5對(duì)細(xì)胞內(nèi)MAPK信號(hào)通路相關(guān)蛋白的影響。結(jié)果顯示，TI5可促進(jìn)ERK的磷酸化。 綜上，TI5可能通過激活ERK/MAPK信號(hào)通路來激活ChAT啟動(dòng)子，其可能是一個(gè)有潛在開發(fā)前景的誘導(dǎo)分化候選藥物。
[Abstract]:At present, the directional differentiation of neural stem cells has become the most attractive topic in life science. With the development of stem cell induction and differentiation technology, stem cell transplantation is possible. Therefore, it has become an important issue in modern medicine and biology to study the mechanism of directional differentiation in vivo and in vitro, to find therapeutic targets and to develop new therapeutic drugs. Acetylcholine is widely found in the central nervous system, in addition to the intestinal nerve and autonomic nerve. Acetylcholine (ache) has been shown to regulate a variety of physiological functions in the central nervous system (CNS), including cognition, attention and arousal, and age-related memory disorders in cholinergic neurons in the central nervous system. A neurodegenerative disease called Alzheimer's disease (AD) is associated with the disease. Acetylcholine transferase (Chat) catalyzes acetylcoenzyme A and choline to produce acetylcholine, which is a rate-limiting enzyme for acetylcholine synthesis of cholinergic neurons. Acetylcholine is stored in presynaptic vesicles, and cystic acetylcholine (VAChT) transports acetylcholine to these vesicles. Acetylcholine binds to two different types of receptors: nicotinic acetylcholine receptor (nAChR) and muscarinic acetylcholine receptor (mAChR). Since ChAT is an important marker of nerve cells, we use the ChAT promoter as the target to establish a screening model and screen the small molecular compound library in order to find the small molecular compounds that can regulate the activity of ChAT promoter. Firstly, we constructed the luciferase report vector of pGL3-ChAT promoter, and transferred the recombinant vector into P19 and HEK293T cells to detect the promoter activity. The results showed that the constructed promoter had high activity. Compared with the control, there was a significant statistical difference between the two groups. Then we used the established luciferase reporter gene method to screen more than 400 small molecular compounds in the laboratory. We found that TI5 could activate the ChAT promoter significantly, and there was a statistically significant difference between the two groups. We verified the regulation of ChAT gene expression of compound TI5 at mRNA level and protein level. In order to elucidate the molecular mechanism of TI5 promoting ChAT expression, we analyzed the intracellular signal transduction pathway of TI5 by transcriptional factor responsive luciferase reporter gene method. It was found that compound TI5 could increase the activity of pSRE-TA-luc luciferase reporter plasmid. So we analyzed the effect of TI5 on intracellular MAPK signaling pathway by Western blotting. The results showed that TI5 could promote the phosphorylation of ERK. TI5 may activate the ChAT promoter by activating the ERK/MAPK signaling pathway, which may be a potential drug candidate for inducing differentiation.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741

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