【作者】 罗星；

【导师】 黄瑾； 杨磊； 张万江； 潘泽民；

【作者基本信息】 石河子大学 ， 生物化学与分子生物学， 2005， 硕士

【摘要】 neuritin是1993年以色列科学家Nedivi等人,从大鼠海马齿状脑回cDNA文库中,发现的一种神经营养因子。研究发现Neuritin蛋白能促进神经突起的生长及其分支和突触的发育成熟,调节突触回路的形成;在神经系统以及神经可塑性相关的区域,neuritin表达水平较高,且神经活动和NTs均能诱导其表达,提示神经活动和NTs促进神经突起生长的作用,可能与neuritin的表达有关。。目的:本研究旨在进一步明确Neuritin蛋白的组织分布、亚细胞定位;并寻找与之相互作用的蛋白,为深入探讨其功能及作用机制奠定理论基础,并为Neuritin在神经系统疾病防治的应用提供理论依据。方法:采用生物信息学手段,分析neuritin基因及蛋白的结构和可能的功能,并进行了基因表达串联分析(Serial Analysis of Gene Expression,SAGE);从人类cDNA文库中扩增出neuritin的开放阅读框,用RT-PCR和免疫组织化学的方法对neuritin进行组织表达谱分析;构建pEGFP-C1-neuritin绿荧光蛋白Neuritin融合表达载体,结合免疫组织化学的方法对neuritin进行亚细胞定位;利用Gal4酵母双杂交系统,以neuritin作诱饵从人胎脑cDNA文库中,筛选与之相互作用的蛋白。结果:1)用生物信息学手段较全面的分析了neuritin核酸及蛋白的结构和可能的功能、组织分布;2)neuritin组织表达谱分析:MTC Panel和免疫组化分析结果均显示:Neuritin蛋白在多种人体正常组织中表达,尤其在脑组织神经节细胞和肝脏中高度表达; 3) Neuritin与GFP的融合蛋白,荧光定位为胞浆,与免疫组化结果一致,即Neuritin蛋白可以在胞浆分布,但不排除膜蛋白的可能。4)酵母双杂交实验:利用Gal4酵母双杂交系统,以neuritin作诱饵从人胎脑cDNA文库中,筛选得到59条与Neuritin蛋白有相互作用的蛋白质的基因,并进行了初步分类和分析。结论:1) MTC Panel组织表达谱实验、多组织免疫组化结果以及SAGE分析均表明:Neuritin蛋白除了脑组织高度表达外,在人体心、肝、肺、小肠以及卵巢等多种正常组织也有表达,提示:Neuritin蛋白在成年期可能不仅仅在中枢神经系统发挥作用。2)通过绿荧光定位和免疫组化的结果证实:Neuritin蛋白为胞浆定位,但不排除膜蛋白的可能。3)酵母双杂交结果显示:Neuritin分别与神经相关、与肿瘤相关以及与信号转导相关的蛋白质有相互作用。提示:Neuritin蛋白可能除了与神经可塑性有关,还可能与细胞增殖以及肿瘤有关。更多还原

【Abstract】 Neuritin was a novel neurotrophic factor which was discovered from the adult rat hippocampus cDNA library by Israel scientist Nedivi in 1993. The expression product of neuritin can enhance the development of neuron axon arbors by promoting synaptogenesis and by increasing the addition of new axon branches, and its expression was induced by neural activity or neurotrophins, which indicated that the neural activities or neurotrophins function were related to neuritin.Objective: The aim of project is to identify the tissue expression profile and intracellular localization of neuritin, and to find proteins that interact with Neuritin using yeast two hybrid system, which provide theoretical basis for further research about its function and mechanism.Method: neuritin structure and its potential function and Serial Analysis of Gene Expression(SAGE) were analyzed using bioinformation technology; neuritin ORF was amplified from human cDNA library, and then neuritin expression profile was showed with RT-PCR and immunohistochemistry; Intracellular localization of neuritin was inferred by immunohistochemistry and eukaryon expression vector combinded with green fluorescence protein, pEGFP-C1-neuritin; Screened a human fetal brain cDNA library with neuritin cDNA as bait using Gal4 yeast two hybrid system.Results:1) Analysis of neuritin structure, expression profile and its potential function with bioinformation technology. 2)neuritin expression profile:multiple Tissure cDNA panel and immunohistochemistry showed that Neuritin expression in a high level in many human normal tissues, especially in ganglion cell of brain and liver. 3) neuritin intracellular location: the result from green fluorescence assay showed Neuritin location in cytoplasm, which was consistent with result of immunohistochemistry. 4)Using yeast two hybrid, screened a human fetal brain cDNA library with neuritin cDNA as bait, and found that the proteins coded by 59 genes had interaction with Neuritin.Conclusion: 1) MTC Panel, multiple tissue immunohistochemistry and SAGE showed that Neuritin was expressed not only in brain, but in heart, liver, lung,intestine and ovary. The results implied that Neuritin play a critical role beyond in nerval system. 2) The results from green fluorescence protein orientation and immunohistochemistry showed that Neuritin was located in cytoplasm. 3. yeast two hybrid results showed that Neuritin interacted with the proteins related to nerve, tumor and signal transduction respectively. These data indicated that Neuritin was possibly relative to cell proliferation and tumorigenesis, as well as to nerves plasticity.更多还原