细胞膜表面雌激素受体标记平台的建立及相关研究

发布时间：2018-02-04 06:03

本文关键词： 雌激素雌激素受体非基因组作用机制细胞膜标记 miRNA　出处：《成都理工大学》2012年硕士论文　论文类型：学位论文

【摘要】：雌激素是一种重要的内源性物质，雌激素在机体内的合成代谢的紊乱会导致脑血管病、骨质疏松病以及乳腺癌，雌激素通过与雌激素受体结合激活细胞内的信号通路从而调节体内的代谢过程。有研究发现，雌激素—受体通路的持续性激活会导致恶性肿瘤的发生。同样，近年来的多项研究发现在许多组织中雌激素可以在数秒或数分钟内快速诱导快速的细胞反应，它可能直接激活了与膜结合的信号通路,这就是雌激素的非基因组作用机制，因此雌激素的非基因组作用机制的研究成为了研究雌激素介导的细胞信号通路的关键。然而如何有效区分mER和胞质内ER成为了目前研究mER非基因组作用的难点和瓶颈，mER的信号终止机制研究方面更是一片空白。而最近的研究又把雌激素与miRNA的协调调控过程联系在一起，因此，本课题采用PPTase酶催化标记膜蛋白的新方法，使其能够将mER与胞内ER区分开来，这对进一步了解mER与雌激素受体之间的信号交流，以及发现新的机制具有重要的意义。而通过构建miRNA细胞模型筛选具有雌激素受体调节作用的化学小分子，对于实时动态地研究mER在不同刺激因素下在细胞膜和细胞内的动态分布也具有重要意义。在细胞膜蛋白标记方法的研究中，针对传统的标记方法的缺陷和不足，如免疫荧光法需要对活细胞进行固定，而GFP融合法不适用于实时动态地研究mER这样在细胞质和细胞核也存在的蛋白质等问题，本文用磷酸泛酰巯基乙胺基转移酶(PPTase)识别特异载体蛋白的方法对细胞膜的mERα作了标记实验，体外实验证明了酶和标记体系均能正常使用，体内标记实验证明了ERα的C端暴露在细胞膜外，并确定了标记条件，为活细胞的标记和观察提供了基础，此外活细胞的标记实验也进一步证明了该标记方法适用于区分细胞膜和细胞质中的雌激素受体蛋白，从而解决了一个关键的科学问题。在化学小分子调控RNAi信号途径的研究中，通过使用化合物库中的天然产物药物对所构建miRNA细胞筛选模型进行筛选，得到了2个效果较好的化合物，实验结果表明，随着化合物浓度的升高，对细胞内蛋白的上调作用也逐渐加强，在最适浓度之后效果呈现减弱趋势，这些化合物为今后实时动态地研究mER在不同刺激因素下在细胞膜和细胞内的动态分布提供了基础。最后，通过对一些结构很有特点的化合物和内源蛋白的结合反应机理进行了相关研究，并首次发现了2个能通过静态猝灭内源蛋白，改变这些蛋白的构象从而与之结合发挥药效的活性小分子，为化学小分子调控细胞膜结合的雌激素受体的筛选提供重要补充。
[Abstract]:Estrogen is an important endogenous substance. The disorder of estrogen biosynthesis in the body can lead to cerebrovascular disease, osteoporosis and breast cancer. Estrogen regulates metabolism by binding to estrogen receptors to activate intracellular signaling pathways. It has been found that continuous activation of estrogen receptor pathways leads to the occurrence of malignant tumors. In recent years, several studies have found that in many tissues, estrogen can induce a rapid cellular response in seconds or minutes, which may directly activate the membrane binding signaling pathway. This is the non-genomic mechanism of estrogen. Therefore, the study of the non-genomic mechanism of estrogen has become the key to study the estrogen-mediated cellular signaling pathway. However, how to effectively distinguish between mER and ER in the cytoplasm has become the current study of non-genomic mER. Difficulties and bottlenecks in use. The study of signal termination mechanism of mER is a blank, and recent studies have linked estrogen with the coordinated regulation of miRNA. In this study, we used a new method of PPTase enzymatic labeling membrane protein to distinguish mER from ER, which can further understand the signal exchange between mER and estrogen receptor. And the discovery of new mechanism is of great significance. By constructing miRNA cell model, we can screen small chemical molecules with estrogen receptor regulation. It is also of great significance to study the dynamic distribution of mER in cell membrane and cell under different stimuli in real time. In the study of cell membrane protein labeling methods, the shortcomings and shortcomings of traditional labeling methods, such as immunofluorescence method need to be fixed on living cells. However, GFP fusion method is not suitable for real-time and dynamic study of proteins such as mER in cytoplasm and nucleus. In this paper, mER 伪 of cell membrane was labeled by the method of identification of specific carrier protein by the method of phospho-2-mercaptoacetyltransferase (PPTase). In vitro, it was proved that the enzyme and the labeling system could be used normally. In vivo labeling experiments demonstrated that the C-terminal of ER 伪 was exposed to the cell membrane, and the labeling conditions were determined, which provided the basis for the labeling and observation of living cells. In addition, the labeling experiments of living cells further proved that this method is suitable for distinguishing estrogen receptor proteins in cell membrane and cytoplasm, thus solving a key scientific problem. In the study of the regulation of RNAi signaling pathway by chemical small molecules, the screening model of miRNA cells was screened by using natural product drugs in the compound library. The results showed that with the increase of the concentration of compounds, the up-regulation of intracellular protein was gradually strengthened, and the effect decreased after the optimal concentration. These compounds provide a basis for real-time and dynamic study of the dynamic distribution of mER in cell membrane and cell under different stimuli. Finally, the binding mechanism of some structural compounds and endogenous proteins was studied, and two endogenous proteins were found to quench by static quenching for the first time. Changing the conformation of these proteins to combine with them to play a drug effect of small molecules provides an important supplement for the chemical small molecules to regulate the screening of estrogen receptors binding to cell membranes.
【学位授予单位】：成都理工大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R346

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