發(fā)布時間：2018-02-04 06:03

  本文關(guān)鍵詞： 雌激素 雌激素受體 非基因組作用機制 細(xì)胞膜標(biāo)記 miRNA　出處：《成都理工大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：雌激素是一種重要的內(nèi)源性物質(zhì)，雌激素在機體內(nèi)的合成代謝的紊亂會導(dǎo)致腦血管病、骨質(zhì)疏松病以及乳腺癌，雌激素通過與雌激素受體結(jié)合激活細(xì)胞內(nèi)的信號通路從而調(diào)節(jié)體內(nèi)的代謝過程。有研究發(fā)現(xiàn)，雌激素—受體通路的持續(xù)性激活會導(dǎo)致惡性腫瘤的發(fā)生。同樣，近年來的多項研究發(fā)現(xiàn)在許多組織中雌激素可以在數(shù)秒或數(shù)分鐘內(nèi)快速誘導(dǎo)快速的細(xì)胞反應(yīng)，它可能直接激活了與膜結(jié)合的信號通路,這就是雌激素的非基因組作用機制，因此雌激素的非基因組作用機制的研究成為了研究雌激素介導(dǎo)的細(xì)胞信號通路的關(guān)鍵。然而如何有效區(qū)分mER和胞質(zhì)內(nèi)ER成為了目前研究mER非基因組作用的難點和瓶頸，mER的信號終止機制研究方面更是一片空白。而最近的研究又把雌激素與miRNA的協(xié)調(diào)調(diào)控過程聯(lián)系在一起，因此，本課題采用PPTase酶催化標(biāo)記膜蛋白的新方法，使其能夠?qū)ER與胞內(nèi)ER區(qū)分開來，這對進一步了解mER與雌激素受體之間的信號交流，以及發(fā)現(xiàn)新的機制具有重要的意義。而通過構(gòu)建miRNA細(xì)胞模型篩選具有雌激素受體調(diào)節(jié)作用的化學(xué)小分子，對于實時動態(tài)地研究mER在不同刺激因素下在細(xì)胞膜和細(xì)胞內(nèi)的動態(tài)分布也具有重要意義。 在細(xì)胞膜蛋白標(biāo)記方法的研究中，針對傳統(tǒng)的標(biāo)記方法的缺陷和不足，如免疫熒光法需要對活細(xì)胞進行固定，而GFP融合法不適用于實時動態(tài)地研究mER這樣在細(xì)胞質(zhì)和細(xì)胞核也存在的蛋白質(zhì)等問題，本文用磷酸泛酰巰基乙胺基轉(zhuǎn)移酶(PPTase)識別特異載體蛋白的方法對細(xì)胞膜的mERα作了標(biāo)記實驗，體外實驗證明了酶和標(biāo)記體系均能正常使用，體內(nèi)標(biāo)記實驗證明了ERα的C端暴露在細(xì)胞膜外，并確定了標(biāo)記條件，為活細(xì)胞的標(biāo)記和觀察提供了基礎(chǔ)，此外活細(xì)胞的標(biāo)記實驗也進一步證明了該標(biāo)記方法適用于區(qū)分細(xì)胞膜和細(xì)胞質(zhì)中的雌激素受體蛋白，從而解決了一個關(guān)鍵的科學(xué)問題。 在化學(xué)小分子調(diào)控RNAi信號途徑的研究中，通過使用化合物庫中的天然產(chǎn)物藥物對所構(gòu)建miRNA細(xì)胞篩選模型進行篩選，得到了2個效果較好的化合物，實驗結(jié)果表明，隨著化合物濃度的升高，對細(xì)胞內(nèi)蛋白的上調(diào)作用也逐漸加強，在最適濃度之后效果呈現(xiàn)減弱趨勢，這些化合物為今后實時動態(tài)地研究mER在不同刺激因素下在細(xì)胞膜和細(xì)胞內(nèi)的動態(tài)分布提供了基礎(chǔ)。 最后，通過對一些結(jié)構(gòu)很有特點的化合物和內(nèi)源蛋白的結(jié)合反應(yīng)機理進行了相關(guān)研究，并首次發(fā)現(xiàn)了2個能通過靜態(tài)猝滅內(nèi)源蛋白，改變這些蛋白的構(gòu)象從而與之結(jié)合發(fā)揮藥效的活性小分子，為化學(xué)小分子調(diào)控細(xì)胞膜結(jié)合的雌激素受體的篩選提供重要補充。
[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 偽 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 偽 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.
【學(xué)位授予單位】：成都理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R346

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