本文選題：砷化物 + p53��； 參考：《廣西醫(yī)科大學(xué)》2017年碩士論文

【摘要】：研究背景及內(nèi)容砷是一種廣泛存在于自然界的重金屬類毒性元素,對(duì)人類健康具有極大危害。通常,高劑量砷化物暴露主要引發(fā)以“細(xì)胞凋亡”等為主要特征的急性毒性損傷效應(yīng);而低劑量砷化物長(zhǎng)期暴露主要引發(fā)以“細(xì)胞癌變”等為主要特征的慢性毒性損傷效應(yīng)。因此,砷化物的健康危害效應(yīng)機(jī)制研究具有重要的基礎(chǔ)理論意義和醫(yī)學(xué)應(yīng)用價(jià)值。本課題組長(zhǎng)期開(kāi)展砷化物誘導(dǎo)細(xì)胞凋亡反應(yīng)的急性毒性損傷效應(yīng)機(jī)制研究工作,在以往研究中獲得了大量具有價(jià)值的研究發(fā)現(xiàn),為砷化物的健康危害評(píng)估和損傷防控策略研究提供了重要的理論依據(jù)和干預(yù)靶標(biāo)。在近期工作中,我們又發(fā)現(xiàn)了砷化物誘導(dǎo)促細(xì)胞凋亡反應(yīng)中伴隨有IKK激酶的兩個(gè)催化亞基——IKKα和IKKβ的表達(dá)水平下調(diào)現(xiàn)象,并且這一現(xiàn)象的發(fā)生是砷化物誘導(dǎo)細(xì)胞走向凋亡的重要前提。在對(duì)以上現(xiàn)象的分子機(jī)制進(jìn)行深入研究過(guò)程中,我們先后發(fā)現(xiàn)了砷化物刺激可通過(guò)激活p53并進(jìn)一步促發(fā)細(xì)胞自噬反應(yīng)從而誘導(dǎo)IKKα進(jìn)入自噬途徑降解,同時(shí)p53也可通過(guò)誘導(dǎo)靶基因ETS-1表達(dá)而協(xié)同介導(dǎo)對(duì)IKKβ的轉(zhuǎn)錄抑制作用。本課題以上述研究結(jié)果為基礎(chǔ),在砷化物處理的HepG2細(xì)胞中,探討了p53信號(hào)通路調(diào)節(jié)砷化物誘導(dǎo)的IKKα自噬降解反應(yīng)和IKKβ轉(zhuǎn)錄抑制的信號(hào)傳遞機(jī)制(見(jiàn)前言部分附圖)。結(jié)果在第一部分工作中,我們首先篩選了能夠介導(dǎo)砷化物促發(fā)IKKα自噬降解反應(yīng)的p53下游靶基因。結(jié)果發(fā)現(xiàn):砷化物刺激HepG2細(xì)胞后能夠誘導(dǎo)自噬相關(guān)p53靶基因DRAM1、ISG20L1、DAPK1、TIGAR、SESTRIN2表達(dá),其中DRAM1能夠介導(dǎo)砷化物誘導(dǎo)的IKKα自噬降解反應(yīng);而TIGAR和SESTRIN2與上述反應(yīng)狀態(tài)完全無(wú)關(guān)。ISG20L1和DAPK1能夠介導(dǎo)砷化物刺激誘導(dǎo)的細(xì)胞自噬反應(yīng),但這種自噬反應(yīng)并不能夠介導(dǎo)IKKα降解;而且ISG20L1在砷化物刺激作用下的誘導(dǎo)表達(dá)反應(yīng)也不受控于p53。以上實(shí)驗(yàn)結(jié)果說(shuō)明:DRAM1是能夠介導(dǎo)砷化物促發(fā)IKKα自噬降解反應(yīng)的p53下游靶基因。在第二部分工作中進(jìn)一步分析了砷化物刺激反應(yīng)中負(fù)責(zé)催化p53/DRAM1信號(hào)傳遞途徑誘導(dǎo)活化的上游蛋白激酶。結(jié)果發(fā)現(xiàn):ATR、CHK1、LKB1、AMPKα、PERK均能夠調(diào)節(jié)砷化物刺激作用下p53的轉(zhuǎn)錄激活活性,然而只有CHK1、LKB1是介導(dǎo)DRAM1誘導(dǎo)表達(dá)并進(jìn)一步促發(fā)IKKα自噬降解反應(yīng)的p53上游蛋白激酶。在第三部分工作中,我們初步分析了砷化物促發(fā)IKKα自噬降解反應(yīng)的分子機(jī)制。由于IKKα和IKKβ高度同源,但只有IKKα能夠進(jìn)入自噬途徑實(shí)現(xiàn)降解;因此我們推測(cè):這種自噬反應(yīng)的選擇特異性一方面可能與IKKα本身的分子結(jié)構(gòu)密切相關(guān),另一方面也可能存在有協(xié)同因子參與協(xié)助IKKα的降解反應(yīng)。我們的實(shí)驗(yàn)結(jié)果顯示:砷化物刺激作用下IKKα能夠通過(guò)其激酶結(jié)構(gòu)域與LC3發(fā)生誘導(dǎo)性結(jié)合反應(yīng)從而進(jìn)入自噬降解途徑,而CHK1、LKB1在此過(guò)程中可能發(fā)揮協(xié)同作用。盡管我們?cè)诘谝徊糠止ぷ髦信懦薎SG20L1和DAPK1在介導(dǎo)砷化物促發(fā)IKKα自噬降解反應(yīng)中的作用,但卻意外地發(fā)現(xiàn)了這兩個(gè)信號(hào)蛋白能夠介導(dǎo)IKKβ的轉(zhuǎn)錄抑制反應(yīng)。因此我們?cè)谧詈笠徊糠止ぷ髦蟹治隽薎SG20L1和DAPK1調(diào)節(jié)砷化物刺激誘導(dǎo)IKKβ轉(zhuǎn)錄抑制的分子機(jī)制。結(jié)果發(fā)現(xiàn):DAPK1能夠協(xié)同ISG20L1作為活化p53-ETS-1-IKKβ轉(zhuǎn)錄抑制信號(hào)傳遞途徑的上游蛋白,并進(jìn)而通過(guò)調(diào)控MDM2依賴的GADD45α誘導(dǎo)表達(dá)反應(yīng)而介導(dǎo)砷化物誘導(dǎo)的促細(xì)胞凋亡效應(yīng)。結(jié)論綜上所述,本論文研究結(jié)果初步揭示了砷化物刺激誘發(fā)IKKα選擇性自噬降解反應(yīng)的分子機(jī)制;同時(shí)也進(jìn)一步完善了砷化物刺激誘導(dǎo)IKKβ轉(zhuǎn)錄抑制的信號(hào)傳遞機(jī)制。以上實(shí)驗(yàn)結(jié)果不僅為IKK激酶在細(xì)胞應(yīng)激反應(yīng)中的基因表達(dá)調(diào)控機(jī)制研究提供了創(chuàng)新性研究發(fā)現(xiàn),并且為砷化物的毒性效應(yīng)機(jī)制研究提供了嶄新內(nèi)容。
[Abstract]:Research background and contents of heavy metal arsenic is a toxic element widely exists in the nature, which is harmful to human health. Generally, the high dose of arsenic exposure is mainly caused by acute toxicity injury "apoptosis" as the main feature; while low dose arsenic exposure mainly caused by the effects of chronic toxicity "cancer cells" as the main feature. Therefore, health damage mechanism of arsenic has important theoretical significance and application value. The acute toxicity of medicine based the research group to carry out long-term arsenic induced apoptosis reaction damage study of the mechanism of effects in previous studies were obtained with a large number of the value found, provide a theoretical basis and an important target for intervention health hazard assessment and damage prevention and control strategy research for arsenic. In recent work, we also found The arsenic induced Pro apoptotic reactions with the two catalytic subunit of IKK kinase, the expression level of IKK alpha and IKK beta cut phenomenon, and this phenomenon is an important prerequisite for arsenic induced cell apoptosis. Further study in the process of the molecular mechanism of the above phenomenon, we the discovery of arsenic stimulation can activate p53 and further promote autophagy reaction to induce the autophagy pathway into IKK alpha degradation, while p53 may also co inhibition mediated transcription of IKK beta by inducing the expression of ETS-1 gene. This subject is based on the above research results, in the treatment of arsenic in HepG2 cells, discusses the signal of IKK alpha and IKK beta transcription autophagy degradation reaction of p53 signaling pathway in regulation of arsenic induced inhibition of the transfer mechanism (see preface Figure). Results in the first part, we first Screening can be mediated by arsenic on p53 downstream target genes IKK alpha autophagic degradation reaction. Results showed that arsenic stimulated HepG2 cells can induce autophagy related p53 target genes DRAM1, ISG20L1, DAPK1, TIGAR, SESTRIN2 expression, DRAM1 can IKK a autophagy mediated degradation reaction induced by arsenic while TIGAR and SESTRIN2; and the reaction condition has nothing to do.ISG20L1 and DAPK1 can response to autophagy mediated arsenic induced autophagy, but this reaction is not mediated by IKK and ISG20L1 in alpha degradation; arsenic stimulation induced expression of the reaction is not controlled by the above p53. results show that DRAM1 is can mediate arsenic on p53 downstream target genes IKK alpha autophagic degradation reaction. In the second part in the further analysis of arsenic in the stimuli responsible for catalysis of p53/DRAM1 signaling pathway activation on Tour the protein kinase. The results showed that: ATR, CHK1, LKB1, AMPK alpha, PERK can regulate arsenic stimulates transcription activation activity under the action of p53, but only CHK1, LKB1 mediates DRAM1 induced expression and further promote the development of IKK alpha autophagy degradation reaction of p53 upstream kinase. In the third part of this work. We analyzed the molecular mechanism of arsenic in promoting IKK alpha autophagy degradation reaction. Because of IKK alpha and IKK beta are highly homologous, but only IKK alpha can enter the autophagy pathway to achieve degradation; we hypothesize that the choice of a specific autophagic response may be closely related to the molecular structure and IKK alpha itself, another also there may be synergistic factors to participate in the degradation reaction of IKK alpha. Our experimental results showed that arsenic stimulated by IKK alpha through its kinase domain and LC3 induced binding reaction to enter autophagy degradation. Size, while CHK1 and LKB1 may play a synergistic role in the process. Although we exclude ISG20L1 and DAPK1 in arsenic mediated priming IKK alpha autophagic degradation reaction in the first part, but accidentally discovered the two signal protein transcription mediated IKK beta inhibitory response. Therefore we analyzed ISG20L1 and DAPK1 regulate arsenic induced molecular mechanism of IKK beta transcription inhibition in the last part of this work. The results showed that DAPK1 can cooperate with ISG20L1 as activation of p53-ETS-1-IKK beta signaling pathway upstream of the transcriptional repressor protein, and then through the apoptosis promoting effect of regulation of MDM2 dependent GADD45 expression induced by alpha reaction mediated by arsenic induced. In conclusion, the molecular mechanism of the results of this study revealed arsenic induced IKK alpha selective autophagic degradation reaction; at the same time further end The arsenic induced transcription inhibition of IKK beta signal transmission mechanism. The above results not only provide the regulation mechanism of innovative research found that gene IKK kinase in cell stress response expression, and toxicity mechanism of arsenic with new content.

【學(xué)位授予單位】：廣西醫(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)】：R114

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本文編號(hào)：1761122