三聚体胞内阳离子通道蛋白B参与蜕皮激素介导的钙离子释放与内流

发布时间：2018-03-04 18:03

  本文选题：TRIC-B　切入点：20E　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景、存在的科学问题级研究意义昆虫是世界上品类、个体数量最丰富的动物类群,对人类在农业、医学等领域的生产活动有着巨大影响。围绕对害虫的防治,多以昆虫作为模型研究多种生理生化现象机理。昆虫包括完全变态昆虫,其发育过程经历卵、幼虫、蛹、成虫和不完全变态昆虫,其发育过程经历卵、幼虫变态发育成成虫。蜕皮(molting)和变态(metamorphosis)是昆虫的生长发育中必须经历的两个过程。昆虫蜕皮变态过程主要受蜕皮激素20羟基蜕皮酮(20-hydroxyecdysone,20E)及保幼激素(Juvenile Hormone,JH)调控,其中蜕皮发生由20E起始,蜕皮的性质由JH决定。经典的20E基因组途径认为20E直接进入细胞并启动下游基因的转录,而近年来的研究表明:某些G蛋白偶联受体(G-protein-coupledreceptor,GPCR)存在于细胞膜上,能够响应蜕皮激素信号,并介导细胞内的钙离子浓度升高。静息状态下,细胞内钙离子贮存在细胞内质网(endoplasmic reticulum,ER)中;激素刺激下,细胞内钙离子由内质网上的储存调控型钙离子通道(store-operated calcium channel,SOC)释放到细胞质。SOC是非神经兴奋性细胞中的钙通道,受细胞信号刺激产生三磷酸肌醇(inositol 1,4,5-triphosphate,IP3),IP3 通过 IP3 受体(inositol 1,4,5-triphosphate receptor,IP3R)介导将内质网贮存的钙离子释放到细胞质。细胞外钙离子由受体调控的钙离子通道内流进入细胞质。由于钙离子是带正电荷的阳离子,因而内质网内储存的钙离子释放到细胞质的过程引起内质网膜内外电荷不平衡。细胞内需要其他离子,如钾离子、氯离子和氢离子等作为平衡离子补充钙离子快速释放引起的内质网膜电位失衡(Fink and Veigel 1996;NAGASAWA et al.1984)。三聚体胞内阳离子通道蛋白(Trimeric intracellular cation channel,TRIC)是一种存在于细胞内质网/肌质网(ER/SR)并附着在核膜上的阳离子通道蛋白,在细胞中主要参与内质网钾离子双向运输。TRIC作为双向钾离子通道参与平衡细胞内钙离子释放导致的内质网电位差。TRIC蛋白通道包含两个亚家族:TRIC-A和TRIC-B。其中TRIC-A大量存在于可兴奋性细胞中,响应兰尼碱受体介导的内质网/肌质网钙离子释放;TRIC-B少量但广泛地存在于各种细胞中,响应IP3R介导的内质网钙离子释放。既然20E能够诱导IP3R开放,而TRIC-B又可以响应IP3R介导的钙离子释放,那么TRIC-B是否参与蜕皮激素20E信号途径?研究报道TRIC-B通道参与发育过程(Yamazaki et al.2009),其机制如何?目前尚无研究报道。本文以鳞翅目害虫棉铃虫(Halicoverpa armigera)作为研究材料,并利用实验室前期建立的棉铃虫表皮细胞系(HaEpi)为平台,研究了 TRIC-B参与调节20E诱导的钙离子释放和内流过程,及其对昆虫发育的影响,并在20E在生长过程中对钙离子的调节机理方面有所促进,为防治害虫提供了新的靶标。研究结果及结论本实验以棉铃虫为实验对象,通过分子生物学技术、生物化学及细胞生物学技术,研究三聚体胞内阳离子通道蛋白B(TRIC-B)在昆虫生长发育过程中的作用,并取得以下结果:TRIC-B在蜕皮期高表达;干扰TRIC-B导致幼虫发育失败死亡;TRIC-B通过维持内质网电荷平衡介导20E诱导的细胞内钙释放和外钙内流;TRIC-B定位于细胞质;干扰TRIC-B导致HaEpi细胞死亡。结论:TRIC-B作为离子平衡通道介导20E调控的内质网中钙离子的有效释放及细胞外钙离子内流。TRIC-B在棉铃虫发育过程和细胞存活中起关键性作用,缺失TRIC-B幼虫和细胞发生死亡。研究的创新点及意义TRIC-B能够维持20E诱导的钙离子释放及细胞外钙离子内流过程。丰富了蜕皮激素20E非基因组途径的理论,验证了 TRIC-B通道在生长发育过程中的重要作用,为害虫控制提供了良好靶标。
[Abstract]:The research background, research significance of insect science problem is the existence of the world category, animal taxa were most abundant in the number of individuals, to human beings in agriculture, medicine and other fields have a huge impact in the production activities. The control of pests, to study a variety of insects as a physiological phenomenon. The mechanism model of insects including holometabolous insects the developmental process of experience, egg, larva, pupa, adult and incomplete metamorphosis development experience, eggs, metamorphosis in larvae develop into adult worms. Molting and metamorphosis (molting) (metamorphosis) is the two process of insect growth and development must be experienced. Molt and metamorphosis process is mainly affected by molting hormone 20 HYDROXYECDYSONE (20-hydroxyecdysone, 20E) and juvenile hormone (Juvenile, Hormone, JH) regulation, which occurs by molting 20E initiation, molting properties determined by the JH. The 20E gene group pathway that 20E directly into the fine The cell and activate its downstream gene transcription, but recent studies have shown that certain G protein coupled receptors (G-protein-coupledreceptor, GPCR) on the cell membrane, in response to ecdysone signal, and mediated by the intracellular calcium concentration increased. The resting state, intracellular calcium stored in the endoplasmic reticulum (endoplasmic reticulum. ER); hormone stimulation, intracellular calcium regulation by storage type calcium channel Er (store-operated calcium channel, SOC).SOC is released into the cytoplasm calcium channel non nerve excitability in the cells by cell signaling stimulates the production of inositol phosphate (inositol three 1,4,5-triphosphate, IP3), IP3 (via IP3 receptor inositol 1,4,5-triphosphate receptor, IP3R) mediated endoplasmic reticulum calcium storage and release into the cytoplasm. Extracellular calcium by calcium channel receptor regulation within the flow into the fine Due to the cytoplasm. The calcium ion is positively charged cations, Ca2 + and ER storage is released into the cytoplasm of process induced endoplasmic reticulum and charge imbalance. Cells need other ions, such as potassium, calcium ion balance as of the rapid release of endoplasmic reticulum membrane potential caused by the imbalance of chloride ion and hydrogen ion (such as Fink and Veigel 1996; NAGASAWA et al.1984). The trimer of intracellular cation channel protein (Trimeric intracellular cation channel, TRIC) is a present in the endoplasmic reticulum / sarcoplasmic reticulum (ER/SR) and attached cation channel protein in the nuclear membrane, the cells mainly involved in endoplasmic reticulum potassium ion transport bidirectional.TRIC as the two potassium channels involved in balance of intracellular Ca2 + release of endoplasmic reticulum potential difference in the protein.TRIC pathway contains two subfamilies: TRIC-A and TRIC-B. which can exist in the TRIC-A Excitatory cells in response to endoplasmic reticulum / ryanodine receptor mediated sarcoplasmic reticulum calcium release; a small amount of TRIC-B but is widely used in all kinds of cells in response to endoplasmic reticulum calcium mediated release of IP3R. Since 20E can induce the opening of IP3R, while TRIC-B can respond to calcium mediated release of IP3R. That TRIC-B is involved in ecdysone signal pathway of 20E research reports? TRIC-B channels are involved in the development process (Yamazaki et al.2009), what is the mechanism? There is no research. This paper reports to Lepidoptera pests of cotton bollworm (Halicoverpa armigera) as the research materials, and the use of cotton bollworm epidermal cell lines the pre established (HaEpi) platform. Study of TRIC-B involved in the regulation of calcium release induced by 20E and flow process, and its effect on insect development, and in the 20E in the process of growth regulating mechanism of calcium ions on surface was enhanced, in order to prevent Provide a new target for treatment of pests. The results and conclusions of this experiment to cotton bollworm as the experimental object, through the technology of molecular biology, biochemistry and cell biology technology research, trimeric intracellular cation channel protein B (TRIC-B) in the process of growth and development of insects in the role, and achieved the following results: TRIC-B in high molting period the expression of TRIC-B in larval development; interference failure death; TRIC-B by maintaining the endoplasmic reticulum charge balance mediated 20E induced intracellular calcium release and calcium influx; TRIC-B located in the cytoplasm; TRIC-B interference induced HaEpi cell death. Conclusion: in the process of the development of cotton bollworm and plays a key role in cell survival.TRIC-B release and extracellular calcium ion balance as TRIC-B channel mediated regulation of 20E in the endoplasmic reticulum in the death of larvae and deletion of TRIC-B cells. The innovation of research and the significance of TRIC-B The process flow of calcium release and extracellular calcium to maintain 20E induced. Enriched 20E ecdysone non genomic signaling theory, verifies the important role of TRIC-B channels in the process of development and growth, provide a good target for pest control.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S433
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本文编号：1566713