利用秀丽线虫研究脑红蛋白的抗氧化损伤作用及细胞骨架蛋白的缺氧应答机制

发布时间：2018-04-14 03:29

本文选题：秀丽线虫 + 脑红蛋白　；参考：《安徽医科大学》2011年硕士论文

【摘要】：秀丽线虫(Caenorhabditis elegans,C. elegans)是1974年Brenner首次公开报道的模式生物,其优点很多,是进行遗传、基因研究方面的首选模式生物。线虫生长周期短,实验室饲养简便,具备完整的发育和解剖学特征,已完成全基因组测序,基因操作便捷,如转基因、基因突变、RNA干涉等。本研究将线虫应用于Ngb对氧化损伤的作用研究及缺氧状态下细胞骨架的变化分析,旨在促进对低氧应答机制的理解及氧化应激损伤保护的防护基础研究。 1、利用秀丽线虫研究Ngb抗氧化损伤的作用Ngb是新近发现的氧结合珠蛋白超家族成员,表达于神经系统,具有抗氧化损伤作用。Ngb含有两种重要的氨基酸:组氨酸和半胱氨酸,对Ngb的蛋白质结构及功能具有重要作用。为了探讨Ngb的抗氧化作用及作用机制,我们使用hNgb转基因线虫来进行百草枯(paraquat,PQ)处理,结果显示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,但未见明显结果,有可能Ngb是通过自身的重要结构来发挥抗氧化作用。我们还构建了Ngb的双突变表达载体,进行显微注射,以期获得Ngb的突变株系,进一步探讨Ngb抗氧化损伤的作用机制。秀丽线虫体内有33种globin,远多于脊椎动物,其中glb-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。为了探讨glb-13的抗氧化作用及其发挥作用的信号通路,我们通过线虫的交配构建了线虫重要通路hif-1与daf-16和glb-13的双突变株系,并进行PQ处理来统计成虫率,结果显示glb-13(tm2825)突变株系不能恢复PQ引起的氧化损伤,hif-1的glb-13双突变株系的抗氧化作用低于glb-13(tm2825),说明hif-1在glb-13抗氧化作用的信号通路上可能发挥重要作用。在mRNA水平上,glb-13表达水平上调,与其抗氧化表型吻合,glb-13具有抗氧化应激作用,而glb-13的抗氧化应激作用不受HIF-1调控,可能与DAF-16和Nrf-2相关。同时,我们构建了以glb-13为启动子的表达载体,通过EGFP的可视化显示glb-13在线虫体内的分布与表达,以期获得其转基因株系,从而从蛋白质水平探讨glb-13的抗氧化作用。从Ngb的同源物角度进行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的功能对于理解Ngb的功能进化性具有重要意义。 2、利用秀丽线虫研究细胞骨架蛋白在缺氧中的作用机制细胞骨架蛋白是支撑细胞的重要结构,不可或缺。细胞骨架蛋白的功能非常广泛且十分重要,参与细胞的基本生命活动,如细胞运动、物质运输和能量转换、细胞分化、信号转导等。其中,细胞骨架在抗缺氧方面的作用同样重要。为了探讨细胞骨架蛋白在缺氧中发挥的作用,选取原肌球蛋白(tropomyosin)突变株系lev-11(x12)研究细胞骨架在缺氧中的作用。结果显示lev-11突变后对缺氧敏感,蛋白水平显示LEV-11上调,说明细胞骨架蛋白能对抗缺氧引起的损伤。
[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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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