Th17细胞在免疫炎症性心肌纤维化中的作用

衰老：一年生贡氏假鳃鳉载脂蛋白E基因的克隆与表达 衰老论文:一年生贡氏假鳃鳉载脂蛋白E基因的克隆与表达 【中文摘要】近年来,与衰老相关的研究越来越成为当今生物研究的热点。寿命的决定因素是多方面的,涉及到复杂的过程,以至于其中大部分的机制还未完全理解。主要原因是由于研究的模式动物的生命周期长,不利于研究等,严重阻碍了研究衰老的进程。由于近年来,对鱼类的衰老研究表明,哺乳类衰老相关的标记也适用于鱼类,如脂褐素(lipofuscin)、β-半乳糖苷酶(β-galatosidase)和神经退变(neurodegeneration),均会随年(月)龄的增加而上升;人类衰老相关基因,如IGF-1R、MTP、p66shc和SIRT1等也被证实在鱼类中存在。随后,研究者发现了新的衰老模式生物一年生假鳃鳉属。本实验室已有成熟的饲养贡氏假鳃鳉的实验条件,而且贡氏假鳃鳉寿命也只有一年左右,便于进行相关衰老方面的研究。载脂蛋白E(Apolipoprotein E,ApoE)是载脂蛋白家族中一员,是血浆极低密度脂蛋白(LDL)、高密度脂蛋白(HDL)、乳糜微粒(CM1)等脂蛋白的组分,能够与低密度脂蛋白受体结合,在血浆胆固醇和甘油三脂的代谢中发挥作用,近些年研究发现载脂蛋白E与阿尔茨海(Alzheimerdisease,AD)、脑血管疾病、糖尿病的并发症等多种疾病相关,载脂蛋白E基因被认为是衰老基因的一种。本文首先成功分离克隆出贡氏假鳃鳉载脂蛋白E基因序列全长,并对其进行序列。分析结果表明该序列全长1018bp,最大开放阅读框798bp,编码262个氨基酸,其中包括N端18个氨基酸组成的信号肽,C端196个氨基酸组成的载脂蛋白结构域,是一种富含碱性 氨基酸的的碱性蛋白。成熟蛋白是由245个氨基酸组成的多肽链,其中包括33个密码子块(33-codon block)和多个由11个或22个氨基酸组成的重复单位,这些重复单位可能是形成二级结构α-螺旋所必需的。具有相当保守的受体位点区域,且也富含碱性氨基酸。这些都与其他物种的载脂蛋白E结构极为相似。同源分析结果表明,贡氏假鳃鳉载脂蛋白E的氨基酸序列与红鳍东方魨载脂蛋白E-1和白斑狗鱼载脂蛋白E相似性分别达到60.7%和48.8%,与爪蟾、鼠类和人类的相似性分别为30.4%、27%和26.2%。该蛋白与其他硬骨鱼的载脂蛋白E同源性很高,与爪蟾和哺乳类同源性偏低,这也与其他研究结果相一致。系统进化分析结果表明,贡氏假鳃鳉载脂蛋白E和红鳍东方魨载脂蛋白E-1独聚一支,而与其他硬骨鱼中载脂蛋白E-1聚为一大簇。推测该蛋白可能属于载脂蛋白E-1家族。其次,利用实时定量PCR(Real-time Quantity PCR)技术对该蛋白的组织表达情况做了研究。分析结果显示,贡氏假鳃鳉载脂蛋白E只在肝脏和心脏中表达,在肝脏中表达极其显著,这与在人类中的研究结果基本一致,人血浆的载脂蛋白E约70%是由肝脏合成分泌的。对不同月龄贡氏假鳃鳉该基因的表达的研究表明,该基因的表达量随着月龄的增长而随之减少。这也与人类衰老相关标记的变化趋势相一致。由此可见,贡氏假鳃鳉载脂蛋白E可以作为贡氏假鳃鳉的衰老标记,为今后利用其进行衰老相关研究奠定基础。综上所述,贡氏假鳃鳉是一种很好的衰老模式动物,其载脂蛋白E可以作为一种衰老标记,为今后研究衰老的分子机制提供了有利的工具和手段。 【英文摘要】In recent years, the issue of aging has been more and more attractive to researchers. The determinants of life span are various and complex, most of which have not been completely understood yet. Some advances have been made in recent years in understanding molecular mechanisms that influence aging and lifespan. However, to some extent, genetic and pharmacological researches on aging are still impeded by the relatively long lifespan of available vertebrate models. Previous study informed us that lipofuscin, senescence associatedβ-galactosidase and neurodegeneration in teleost species express in an age-dependent manner. Besides, the precocious expression of those phenotypic markers above coincide with the notion that short life span in N.furzeri is due to accelerated aging. We started a aging related study on Nothobranchius guentheri, a species kept in our laboratory with a maximum life span of one year, which might represent a very useful model for comparative genomics of aging. The components of plasma lipoproteins are known as apolipoproteins, which are mainly synthesized in the liver and intestine. Apolipoproteins bind to lipids and play important roles in lipid transportation and lipid admission in the circulation system. In human, the major physiological roles of ApoE are mediating the cellular recognition and internalization of lipoproteins with members of the low-density lipoprotein receptor. ApoE was also found in the peripheral and central nervous systems. ApoE as a aging gene may play a role in several diseases, such as Alzheimer’ s disease, gardiac and vasular diseases and diabetes Mellitus Complications.In this study, a full-length cDNA for ApoE, denominated NgApoE, was cloned from the N. guentheri. This cDNA sequence is 1018bp in length, and codes for a polypeptide of 262 amino acid residues, which contains a signal peptide with 18 amino acid residues. Mature NgApoE polypeptide consists of 245 amino acid residues, including a 33-codon block and tandem repeat units of 11 or 22 amino acid residues predicted to form amphipathicα-helical secondary structures. The deduced protein has the complete conserved domain of Apolipoprotein. Comparative analysis of the amino acid sequence in NgApoE and other vertebrates revealed that NgApoE showed higher levels of identity with their corresponding orthologs from teleosts than from mammals. NgApoE is 60.7% and 48.8% identical to ApoE-1 of Fugu rubripes and ApoE of Northern pike; and shows 30.4%, 27% and 26.2% identity to frog, mouse and human, respectively. The overall secondary structures of ApoE were also generally conserved among vertebrates, despite of modest or low levels of amino acid sequence identity in different species. The phylogenetic tree showed that NgApoE formed an independent cluster with Fugu rubripes ApoE-1. In the teleost group, three ApoE-1s and one ApoE clustered together while three ApoE-2s and other ApoEs are grouped in another clade. The present study characterizes an apoE gene homologous to mammalian apoEs from the Redtail notho. Consequently, the NgApoE may be ApoE-1 .The tissue localization of the ApoE has been studied in some species of teleost. In this study, Real-time Quantity PCR showed that the transcripts are specifically expressed in liver, with weak expression in heart. This is consistent with the results in human, which indicate that two-thirds to three-quarters ApoE of plasma derive from the liver secretion. Our study on relationship between age and expression in N. guentheri shows that ApoE gene expression decreases with aggravating aging process. The trend of the expression of ApoE is also similar to that of human aging-related markers. So we could preliminarily predict that ApoE can serve as an aging marker in N. guentheri, which will possibly be useful for aging reaseach.In conclusion, ApoE can be used as a kind of aging phenotypic marker in N. guentheri. While N. guentheri is a very useful model for comparative genomics of aging, in which many molecular mechanism of aging gene can be explored,ApoE will undoubtedly serve to enhance the reseach on aging. 【关键词】衰老 贡氏假腮鳉 载脂蛋白 E RACE 实时定量PCR 【英文关键词】aging Nothobranchius guentheri ApoE RACE Real-time Quantity PCR 【备注】索购全文在线加我:1.3.99.3.8848 同时提供论文一对一写作指导和论文发表委托服务 【目录】一年生贡氏假鳃鳉载脂蛋白E基因的克隆与表达 摘 要 5-7 Abstract 7-9 第一章 文献综述 11-26 0 前言 11-12 1 贡氏假鳃鳉概况 12-13 2 假鳃鳉研究进 展 13-15 3 载脂蛋白E 概述 15-25 4 本文的研究内容 与意义 25-26 5 技术路线 26 第二章 贡氏假鳃鳉基因 的克隆与序列分析 26-54 0 前言 26-27 1 与 27-42 2 结果 42-53 3 讨论 53-54 第三章 贡氏 假鳃鳉载脂蛋白E 基因的时空表达研究 54-65 0 前言 54 1 材料和方法 54-58 2 结果 58-63 3. 讨论 63-65 参考文献 65-76 缩略语 76-77 致谢 77-78 78