本文選題：角化不良蛋白　切入點(diǎn)：誘導(dǎo)凋亡　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：端粒酶的上調(diào)和激活在大多數(shù)腫瘤類別中被發(fā)現(xiàn)并且可以誘導(dǎo)正常細(xì)胞的惡性轉(zhuǎn)化,其在腫瘤進(jìn)展中的重要作用受到廣泛關(guān)注。端粒酶由端粒酶逆轉(zhuǎn)錄酶TERT、端粒酶RNA組分TERC和其他結(jié)合蛋白組成,他們共同參與端粒酶復(fù)合體的活化,在這些結(jié)合蛋白中的Dyskerin扮演了一個(gè)關(guān)鍵角色。Dyskerin的過表達(dá)已被證實(shí)在多種腫瘤進(jìn)程中起到重要作用并與不良預(yù)后有關(guān)。目前的研究表明Dyskerin同時(shí)影響核糖體生物合成和端粒酶復(fù)合體的穩(wěn)定。一方面Dyskerin活性的減低降低TERC水平并誘導(dǎo)端�？s短;另一方面,Dyskerin功能的缺失會(huì)改變抗凋亡因子比如bcl-2和bcl-xl的信使RNA的轉(zhuǎn)錄和修飾。這些生物效應(yīng)最終導(dǎo)致細(xì)胞周期阻滯和細(xì)胞凋亡。另外,還有其他相關(guān)研究指出該基因在核質(zhì)穿梭、DNA損傷修復(fù)和細(xì)胞粘附中扮演重要角色�；贒yskerin同時(shí)在細(xì)胞和機(jī)體的生理進(jìn)程中起到的重要作用,靶向這些生理進(jìn)程已經(jīng)被探索作為新藥開發(fā)的作用靶點(diǎn),以期望提高選擇性并優(yōu)先殺死腫瘤細(xì)胞。目的:基于國(guó)內(nèi)外研究和實(shí)驗(yàn)室經(jīng)驗(yàn)積累,借助計(jì)算機(jī)輔助藥物設(shè)計(jì)技術(shù),設(shè)計(jì)合成靶向Dyskerin的色酮小分子化合物并進(jìn)行活性評(píng)價(jià)和初步的作用機(jī)制研究。內(nèi)容和方法:1、基于Dyskerin的蛋白結(jié)構(gòu)分析其可能的活性位點(diǎn),通過分子對(duì)接和虛擬篩選,遴選出高活性的目的蛋白小分子抑制劑的骨架結(jié)構(gòu),并通過化學(xué)合成的手段合成出一系列的目標(biāo)化合物。2、對(duì)合成出的一系列化合物進(jìn)行抗腫瘤活性評(píng)價(jià),以篩選出高活性低毒性的小分子化合物。主要方法有細(xì)胞增殖實(shí)驗(yàn)(MTT法)、流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡(Annexin V-FITC/PI雙染)、檢測(cè)線粒體膜電位MPP(JC-1法)、檢測(cè)細(xì)胞周期(PI單染)。3、挑選高活性的小分子化合物進(jìn)行初步的作用機(jī)制研究�；诨钚栽u(píng)價(jià)的相關(guān)結(jié)果和目標(biāo)靶點(diǎn)的功能,通過蛋白免疫印跡實(shí)驗(yàn)檢測(cè)其凋亡和周期阻滯相關(guān)蛋白,探尋其誘導(dǎo)腫瘤細(xì)胞凋亡的通路機(jī)制。4、新型小分子化合物的靶點(diǎn)研究。研究小分子化合物是否通過抑制設(shè)想的靶蛋白發(fā)揮作用。主要方法是檢測(cè)目標(biāo)蛋白及其結(jié)合蛋白的表達(dá)和調(diào)控情況并通過免疫熒光技術(shù)進(jìn)行目的蛋白的定位5、TRAP-PCR-ELISA法檢測(cè)小分子化合物對(duì)腫瘤細(xì)胞端粒酶活性的抑制作用。結(jié)果:借助計(jì)算機(jī)輔助藥物設(shè)計(jì)技術(shù)進(jìn)行結(jié)構(gòu)設(shè)計(jì),用化學(xué)合成的手段合成了一系列的小分子化合物,通過活性評(píng)價(jià)得到數(shù)個(gè)高抗腫瘤活性低毒性的色酮小分子化合物。其中化合物8的效果較好,我們挑選其進(jìn)行了靶點(diǎn)和作用機(jī)制研究。實(shí)驗(yàn)結(jié)果表明,化合物8能顯著降低細(xì)胞活力,并誘導(dǎo)胃癌細(xì)胞凋亡。其誘導(dǎo)胃癌細(xì)胞凋亡的機(jī)制是通過阻滯細(xì)胞周期于G2/M期、破壞線粒體功能、引起嚴(yán)重的DNA損傷等途徑實(shí)現(xiàn)的。靶點(diǎn)研究表明,化合物8作用于胃癌細(xì)胞可以顯著抑制目的蛋白Dyskerin的表達(dá)并同時(shí)抑制h TERT蛋白,導(dǎo)致端粒酶活性的顯著下降。以上結(jié)果表明化合物8是一個(gè)高效的Dyskerin抑制劑并具有優(yōu)越的體外抗腫瘤活性。結(jié)論:Dyskerin是一個(gè)很有潛力的藥物作用靶點(diǎn),靶向Dyskerin是抗腫瘤新藥開發(fā)中的一個(gè)成功的新嘗試�；衔�8作為Dyskerin抑制劑是一個(gè)很有潛力的抗腫瘤新藥。
[Abstract]:Telomerase upregulation and activation in most tumor types were found and can induce malignant transformation of normal cells, it plays an important role in tumor progression concern. Telomerase by telomerase reverse transcriptase TERT, telomerase RNA component TERC and other binding proteins, they participate in the activation of telomerase, in these binding proteins Dyskerin played over expression has been shown to play an important role in a variety of tumors and is associated with poor prognosis in the process of a key role in.Dyskerin. The present study shows that Dyskerin also affects the stability of ribosomal biosynthesis and telomerase complex. On the one hand to reduce the activity of Dyskerin and decrease the level of TERC induced by telomere shortening; on the other hand, the lack of the Dyskerin function will change the transcription and modification of anti apoptotic factors such as Bcl-2 and Bcl-xL messenger RNA. These biological effects. Should ultimately lead to cell cycle arrest and apoptosis. In addition, there are other related research points out that the shuttle gene in the nucleoplasm, DNA damage repair and cell adhesion plays an important role. At the same time, the important role of Dyskerin in the physiological process of cells and organisms plays based on targeting these physiological process has been explored as a target for new drugs the development, in order to improve the selectivity and preferentially kill tumor cells. Objective: Based on domestic and foreign research and laboratory experience, with the help of computer aided drug design technology, design and synthesis of small molecules targeting chromone compounds Dyskerin and study the activity evaluation and preliminary mechanism. Methods and contents: 1, protein structure analysis of Dyskerin the possible active sites based on through molecular docking and virtual screening, selection of skeleton protein inhibitors to high activity of the structure, and Chemical synthesis method of synthesis of a series of target compounds.2, anti-tumor activity evaluation of a series of compounds were synthesized and screened with small molecular compounds with high activity low toxicity. The main methods of cell proliferation assay (MTT method), cell apoptosis by flow cytometry (Annexin V-FITC/PI staining). Detection of mitochondrial membrane potential MPP (JC-1), to detect the cell cycle (PI staining).3, preliminary study on Mechanism of small molecule compounds were selected with high activity. The activity evaluation and target function based on the apoptosis and cycle arrest related protein was detected by Western blotting experiments to explore the pathway the mechanism of tumor cell apoptosis induced by.4, the research target of novel small molecule compounds. Play the role of small molecule compounds by target protein. The main method is to inhibit the idea of target protein and its detection With the expression and regulation of protein and protein localization by immunofluorescence technique 5, inhibition of TRAP-PCR-ELISA for the detection of small molecule compounds on the activity of telomerase in tumor cells. Results: with the help of computer aided drug design techniques for structural design, a series of small molecular compounds were synthesized by means of chemical synthesis, get a high number of antitumor activity and low toxicity of chromone small molecule compounds through the activity evaluation. The effect of compound 8 is better, we choose the studied targets and mechanisms. The experimental results show that the 8 compounds can significantly reduce cell viability and induce apoptosis of gastric cancer cells. The apoptosis of gastric cancer cells induced by the mechanism is through cell cycle arrest in G2/M, damage to mitochondrial function, caused by DNA damage pathway. Serious target research showed that 8 compounds on the gastric cancer The expression of cell can significantly inhibit Dyskerin protein and inhibit the H TERT protein, resulting in a significant decrease in telomerase activity. These results show that compound 8 is an efficient Dyskerin inhibitor and has superior antitumor activity in vitro. Conclusion: Dyskerin is a potential drug target, target is Dyskerin the success of a new attempt in the development of new anticancer drugs. 8 compounds as Dyskerin inhibitors is a potential anti-tumor drug.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R91

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