调控ChAT基因表达的小分子化合物筛选及其初步机制研究

发布时间：2018-05-19 21:14

本文选题：药物筛选 + 神经干细胞　；参考：《吉林大学》2014年硕士论文

【摘要】：目前，神经干细胞的定向诱导分化研究已经成为生命科学领域中最引人注目的热点。随着干细胞定向诱导分化技术的发展，使得干细胞移植治疗成为可能。那么，在体内外研究定向分化的发生机制、寻找治疗靶点、开发新的治疗药物，已成为现代医学和生物学研究的重点问题。乙酰胆碱广泛存在于中枢神经系统中，除此之外还存在于肠神经与植物神经中。乙酰胆碱(ACh)已被证明在中枢神经系统（CNS）调节着不同的生理功能，包括认知、注意力和觉醒等，在中枢神经系统中胆碱能神经元的机能失调也与年龄相关的记忆障碍、神经系统退行性疾病阿尔茨海默病（AD）的发病相关。乙酰胆碱转移酶（ChAT）催化乙酰辅酶A和胆碱生成乙酰胆碱，是胆碱能神经元递质乙酰胆碱合成的限速酶。乙酰胆碱储存于突触前小泡，囊状乙酰胆碱（VAChT）负责转运加载乙酰胆碱到这些囊泡。乙酰胆碱结合两种不同类型的受体：烟碱乙酰胆碱受体（nAChR）和毒蕈碱型乙酰胆碱受体（mAChRs）。鉴于ChAT是神经细胞重要的标志物，我们以ChAT启动子为靶点建立筛选模型，对小分子化合物库进行筛选，以期发现能够调节ChAT启动子活性的小分子化合物。我们首先构建了pGL3-ChAT启动子的荧光素酶报告载体，并将重组载体转入P19和HEK293T细胞中检测启动子活性，结果显示，构建的启动子具有较高的活性，，与对照相比在统计学上存在极显著性差异（**P㩳0.01）。之后我们利用已建立好的荧光素酶报告基因法对实验室中的400多种小分子化合物进行筛选，发现TI5可明显激活ChAT启动子，与对照相比在统计学上存在极显著性差异（*P㩳0.05），随后，我们从mRNA水平和蛋白质水平对化合物TI5的ChAT基因表达调控作用进行了验证。为了阐明TI5促进ChAT表达的分子机制，我们通过转录因子应答性荧光素酶报告基因法对TI5的细胞内信号转导途径进行了分析，发现化合物TI5能够增加pSRE-TA-luc荧光素酶报告质粒的活性，于是我们应用免疫印迹法分析了TI5对细胞内MAPK信号通路相关蛋白的影响。结果显示，TI5可促进ERK的磷酸化。综上，TI5可能通过激活ERK/MAPK信号通路来激活ChAT启动子，其可能是一个有潜在开发前景的诱导分化候选药物。
[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.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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