本文選題：秀麗線蟲 + 腦紅蛋白��； 參考：《安徽醫(yī)科大學》2011年碩士論文

【摘要】：秀麗線蟲(Caenorhabditis elegans,C. elegans)是1974年Brenner首次公開報道的模式生物,其優(yōu)點很多,是進行遺傳、基因研究方面的首選模式生物。線蟲生長周期短,實驗室飼養(yǎng)簡便,具備完整的發(fā)育和解剖學特征,已完成全基因組測序,基因操作便捷,如轉(zhuǎn)基因、基因突變、RNA干涉等。本研究將線蟲應(yīng)用于Ngb對氧化損傷的作用研究及缺氧狀態(tài)下細胞骨架的變化分析,旨在促進對低氧應(yīng)答機制的理解及氧化應(yīng)激損傷保護的防護基礎(chǔ)研究。 1、利用秀麗線蟲研究Ngb抗氧化損傷的作用Ngb是新近發(fā)現(xiàn)的氧結(jié)合珠蛋白超家族成員,表達于神經(jīng)系統(tǒng),具有抗氧化損傷作用。Ngb含有兩種重要的氨基酸:組氨酸和半胱氨酸,對Ngb的蛋白質(zhì)結(jié)構(gòu)及功能具有重要作用。為了探討Ngb的抗氧化作用及作用機制,我們使用hNgb轉(zhuǎn)基因線蟲來進行百草枯(paraquat,PQ)處理,結(jié)果顯示Ngb具有抗氧化作用。使用免疫共沉淀分析Ngb的相互作用蛋白,但未見明顯結(jié)果,有可能Ngb是通過自身的重要結(jié)構(gòu)來發(fā)揮抗氧化作用。我們還構(gòu)建了Ngb的雙突變表達載體,進行顯微注射,以期獲得Ngb的突變株系,進一步探討Ngb抗氧化損傷的作用機制。 秀麗線蟲體內(nèi)有33種globin,遠多于脊椎動物,其中g(shù)lb-13是人源Ngb(hNgb)的同源物,氨基酸序列具有20%的相似性,有相同的功能域。為了探討glb-13的抗氧化作用及其發(fā)揮作用的信號通路,我們通過線蟲的交配構(gòu)建了線蟲重要通路hif-1與daf-16和glb-13的雙突變株系,并進行PQ處理來統(tǒng)計成蟲率,結(jié)果顯示glb-13(tm2825)突變株系不能恢復(fù)PQ引起的氧化損傷,hif-1的glb-13雙突變株系的抗氧化作用低于glb-13(tm2825),說明hif-1在glb-13抗氧化作用的信號通路上可能發(fā)揮重要作用。在mRNA水平上,glb-13表達水平上調(diào),與其抗氧化表型吻合,glb-13具有抗氧化應(yīng)激作用,而glb-13的抗氧化應(yīng)激作用不受HIF-1調(diào)控,可能與DAF-16和Nrf-2相關(guān)。同時,我們構(gòu)建了以glb-13為啟動子的表達載體,通過EGFP的可視化顯示glb-13在線蟲體內(nèi)的分布與表達,以期獲得其轉(zhuǎn)基因株系,從而從蛋白質(zhì)水平探討glb-13的抗氧化作用。從Ngb的同源物角度進行Ngb的抗氧化作用研究,反映了Ngb具有抗氧化的作用,因此研究glb-13的功能對于理解Ngb的功能進化性具有重要意義。 2、利用秀麗線蟲研究細胞骨架蛋白在缺氧中的作用機制 細胞骨架蛋白是支撐細胞的重要結(jié)構(gòu),不可或缺。細胞骨架蛋白的功能非常廣泛且十分重要,參與細胞的基本生命活動,如細胞運動、物質(zhì)運輸和能量轉(zhuǎn)換、細胞分化、信號轉(zhuǎn)導(dǎo)等。其中,細胞骨架在抗缺氧方面的作用同樣重要。為了探討細胞骨架蛋白在缺氧中發(fā)揮的作用,選取原肌球蛋白(tropomyosin)突變株系lev-11(x12)研究細胞骨架在缺氧中的作用。結(jié)果顯示lev-11突變后對缺氧敏感,蛋白水平顯示LEV-11上調(diào),說明細胞骨架蛋白能對抗缺氧引起的損傷。
[Abstract]:Caenorhabditis eleans C., a model organism first reported by Brenner in 1974, has many advantages and is the preferred model organism for genetic and genetic research.Nematode has short growth cycle, simple laboratory breeding, complete developmental and anatomical characteristics, complete genome sequencing, gene manipulation, such as transgenic, gene mutation RNA interference and so on.In this study, nematodes were applied to study the effects of Ngb on oxidative damage and the changes of cytoskeleton in hypoxic state. The purpose of this study was to promote the understanding of hypoxia response mechanism and the protection of oxidative stress injury.1. Studies on the antioxidant damage of Ngb by nematode. Ngb is a newly discovered member of the oxygen binding globin superfamily, expressed in the nervous system. NGB contains two important amino acids: histidine and cysteine.It plays an important role in the protein structure and function of Ngb.In order to investigate the antioxidation effect and mechanism of Ngb, hNgb transgenic nematode was used to treat paraquat PQs. The results showed that Ngb had antioxidant effect.The interaction proteins of Ngb were analyzed by immunoprecipitation, but the results were not obvious. It is possible that Ngb plays an important role in antioxidation through its own important structure.We also constructed a double mutant expression vector of Ngb and microinjected it in order to obtain the mutant strain of Ngb and to further explore the mechanism of antioxidant damage of Ngb.There are 33 species of globin in nematodes, much more than vertebrates, in which glb-13 is the homologue of human ngbhNgb, and the amino acid sequence has 20% similarity and the same functional domain.In order to investigate the antioxidant effect of glb-13 and its signaling pathway, we constructed the double mutant lines of hif-1, daf-16 and glb-13, which are important pathway of nematode through mating, and used PQ to calculate the adult rate.The results showed that glb-13 tm2825) could not recover the oxidative damage induced by PQ in the glb-13 double mutant line of Glif-1, which was lower than that in the glb-13tm2825 strain, suggesting that hif-1 may play an important role in the signaling pathway of glb-13 antioxidation.The expression of glb-13 was up-regulated at the level of mRNA, which coincided with the antioxidant phenotype of glb-13. However, the antioxidant stress of glb-13 was not regulated by HIF-1, which might be related to DAF-16 and Nrf-2.At the same time, we constructed the expression vector with glb-13 as promoter, and visualized the distribution and expression of glb-13 in vivo by EGFP, in order to obtain its transgenic lines, and to explore the antioxidant effect of glb-13 from protein level.The study on the antioxidant activity of Ngb from the point of view of Ngb homologues shows that Ngb has antioxidant effect, so it is important to study the function of glb-13 in understanding the functional evolution of Ngb.2. To study the mechanism of cytoskeleton protein in hypoxia by using nematode.Cytoskeleton protein is an important supporting cell structure, indispensable.The function of cytoskeleton protein is very extensive and very important. It is involved in the basic life activities of cells, such as cell movement, material transport and energy conversion, cell differentiation, signal transduction and so on.The role of cytoskeleton in anti-hypoxia is also important.In order to investigate the role of cytoskeleton protein in hypoxia, a mutant strain lev-11x12 was selected to study the role of cytoskeleton in hypoxia.The results showed that lev-11 mutation was sensitive to hypoxia and the protein level was up-regulated by LEV-11, suggesting that cytoskeleton protein could antagonize hypoxia induced damage.
【學位授予單位】：安徽醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R363

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