糖原代谢相关基因在中华绒螯蟹免疫应答中的调控作用

发布时间：2018-03-28 06:14

  本文选题：中华绒螯蟹　切入点：糖原合酶激酶3　出处：《中国科学院大学(中国科学院海洋研究所)》2017年硕士论文

【摘要】：免疫系统持续地感知和应答环境的威胁,需要消耗大量的能量,能量缺乏会导致免疫系统抑制和抗性降低,所以免疫反应过程中能量的调节十分关键。PI3K/Akt信号通路过去被认为是重要的代谢调节通路,在糖原代谢中发挥关键作用,近年来的研究发现PI3K/Akt参与了对于炎症的调节。与PI3K/Akt关系紧密的RAS/ERK信号通路亦被证明对炎症的发展起重要的调控作用。本研究以中华绒螯蟹(Eriocheir sinensis)为研究对象,利用分子生物学和生物信息学等手段,研究糖原合成酶激酶3以及其上游的两种调控激酶蛋白激酶B(Akt)与细胞外信号调节激酶2(ERK2),探索PI3K与ERK信号通路在中华绒螯蟹固有免疫反应中所发挥的作用。糖原合成酶激酶-3(GSK3)是催化丝氨酸/苏氨酸的蛋白激酶,最早被鉴定为糖原合成的调控分子。本研究从中华绒螯蟹中克隆了GSK3同源基因(命名为EsGSK3)。其开放阅读框长1824 bp,编码一个具有607个氨基酸的蛋白质。在EsGSK3中存在保守的丝氨酸/苏氨酸激酶结构域和DNA结合结构域。EsGSK3首先与日本沼虾(Macrobrachium nipponense)的GSK3-β聚类后纳入无脊椎动物分支,与来自脊椎动物的GSK3s形成不同的分支。在中华绒螯蟹的肝胰腺、眼柄、肌肉、性腺、血细胞和造血组织中均可检测到EsGSK3的mRNA表达,其中在肝胰腺中的表达水平最高,且EsGSK3蛋白主要定位于血细胞的细胞质中。EsGSK3 mRNA表达水平在嗜水气单胞菌(Aeromonas hydrophila)刺激后6小时显著升高(p0.05),在48小时后逐渐降至初始水平(p0.05)。脂多糖诱导的肿瘤坏死因子(TNF)-α因子(EsLITAF)的mRNA表达水平在嗜水气单胞菌刺激后显著升高。然而,利用特异性抑制剂锂抑制EsGSK3的活性后,中华绒螯蟹肝胰腺中糖原水平显著增加(p0.05),血淋巴上清中葡萄糖的含量显著下调,血淋巴细胞中的EsLITAF mRNA表达水平和血清中TNF-α含量均受到显著抑制,而血淋巴细胞中核因子κB抑制剂(IκB)的mRNA表达水平显著上升(p0.05),血细胞的吞噬率显著升高,过氧化氢酶、碱性磷酸酶活性以及一氧化氮(NO)合成的增强。作为GSK3通路上游的激酶,细胞外信号调节激酶(ERK)被认为是丝裂原活化蛋白激酶(MAPK)信号通路中一系列基因代谢的调节因子。从中华绒螯蟹cDNA文库中鉴定出ERK的同源基因,命名为EsERK。其开放阅读框全长为1746 bp,编码581个氨基酸组成的多肽。分别分析了体内EsERK的mRNA在受嗜水气单胞菌刺激和dsRNA干扰后的表达变化。发现在嗜水气单胞菌刺激6小时后,中华绒螯蟹血淋巴细胞中的EsERK mRNA表达水平显著升高(p0.05),在48小时逐渐降低(p0.05)。对EsERK进行RNA干扰24小时后,中华绒螯蟹肝胰腺中糖原水平显著增加(p0.05),血淋巴上清中葡萄糖的含量显著下调,血淋巴细胞中核因子κB抑制剂(IκB)的mRNA表达水平显著上升(p0.05),超氧化物歧化酶活性显著升高(p0.05)。在抑制EsGSK3活性6小时后,中华绒螯蟹血淋巴细胞中EsIκB的mRNA表达水平显著从中华绒螯蟹cDNA文库中鉴定出蛋白激酶B(Akt)基因,并将其命名为EsAkt。其开放阅读框全长为1395 bp,编码一个具有464个氨基酸的蛋白质,其理论分子量为53.17 kDa,理论等电点为5.83。基于RT-PCR分析,在所有检测的组织,包括血细胞,性腺,肝胰腺,鳃,肌肉和眼柄中均检测到EsERK基因mRNA表达,其中在肌肉的表达水平最高。在受到嗜水气单胞菌(A.hydrophila)刺激12小时后,中华绒螯蟹血淋巴细胞中EsAkt的mRNA表达水平显著升高(p0.05)。以上结果表明,EsGSK3可以通过诱导TNF-α产生,抑制血细胞吞噬作用,并调节免疫相关酶的活性及NO的合成调控中华绒螯蟹的固有免疫应答反应。EsERK可以通过调节IκB和TNF-α转录因子的表达参与免疫应答,抑制它们的作用会对通过PI3K/Akt与Ras/ERK通路导致的炎症反应造成影响。此外EsAkt亦表现出对于菌刺激的响应。本研究首次揭示了GSK3、ERK及Akt作为糖原代谢相关通路的关键调节分子,在调节中华绒螯蟹的固有免疫应答中发挥作用,为深入研究PI3K与ERK信号通路在感染防治及炎症控制中的作用提供了基础。
[Abstract]:The immune system continues to perception and response to environmental threats, need to consume a large amount of energy, energy deficiency leads to a decrease in the suppression of the immune system and resistance, so the energy in the process of regulating the immune response is the key of.PI3K/Akt signaling pathway was regarded as an important metabolic pathway, plays a key role in glycogen metabolism, the study found that in recent years PI3K/Akt is involved in regulating inflammation. RAS/ERK signaling pathway has a close relationship with the PI3K/Akt proved to be an important regulatory role in the development of inflammation. In this study, the Chinese Mitten Crab (Eriocheir sinensis) as the research object, using molecular biology and bioinformatics methods, study of glycogen synthase kinase 3 and its upper reaches two regulation of protein kinase B kinase (Akt) and extracellular signal regulated kinase 2 (ERK2), to explore the PI3K and ERK signaling pathway in innate immunity against Chinese Mitten Crab We should take the role of glycogen synthase kinase. -3 (GSK3) is a serine / threonine protein kinase catalytic, was identified as the molecular regulation of glycogen synthesis. In this study, the homologous genes of GSK3 were cloned from Chinese Mitten Crab (named EsGSK3). The open reading frame of 1824 BP in length, encoding a a protein of 607 amino acids in EsGSK3. The existence of conserved serine / threonine kinase domain and DNA binding domain of.EsGSK3 first and m.nipponense (Macrobrachium nipponense) GSK3- beta after clustering into the invertebrate branch, formed different branches and GSK3s. In the spinal animal from Chinese mitten crab hepatopancreas, eyestalk and muscle, gonad, blood cells and hematopoietic tissue expression can be detected in EsGSK3 mRNA, the expression level in hepatopancreas is the highest, and the cytoplasmic EsGSK3 protein was mainly localized in blood cells in.EsGSK3 mRNA The expression level in Aeromonas hydrophila (Aeromonas hydrophila) 6 hours after stimulation was significantly increased (P0.05), in 48 hours after gradually reduced to the initial level (P0.05). Tumor necrosis factor induced by lipopolysaccharide (TNF) - alpha factor (EsLITAF) expression level of mRNA was significantly increased in Aeromonas hydrophila after stimulation. However, the inhibition of EsGSK3 by specific inhibitors of the activity of lithium, glycogen levels of Chinese mitten crab hepatopancreas significantly increased (P0.05), the content of glucose in haemolymph supernatant significantly reduced blood lymphocytes EsLITAF expression level of mRNA and serum TNF- content was obviously inhibited, while blood lymphocyte nuclear factor kappa the inhibitor of B (I K B) significantly increased the expression level of mRNA (P0.05), significantly increased the phagocytic rate of blood cells catalase, alkaline phosphatase activity and nitric oxide (NO) synthesis is enhanced. As the GSK3 upstream kinase, fine Extracellular signal regulated kinase (ERK) is considered to be the mitogen activated protein kinase (MAPK) signaling pathway in a series of regulatory factor gene metabolism. From Eriocheir sinensis cDNA library identified homologous gene ERK, named EsERK. and its ORF is 1746 BP, encoding a polypeptide of 581 amino acids. Analysis of EsERK in the expression of mRNA in affected by Aeromonas hydrophila stimulation and after dsRNA interference. Found that stimulating 6 hours in Aeromonas hydrophila, the expression level of Chinese mitten crab EsERK mRNA lymphocytes increased significantly (P0.05), decreased in 48 hours (P0.05) of EsERK. RNA interference after 24 hours, the glycogen level of Chinese mitten crab hepatopancreas significantly increased (P0.05), the content of glucose in haemolymph supernatant significantly reduced blood lymphocyte nuclear factor kappa B inhibitor (I K B) significantly increased the expression level of mRNA (P0.05), The activity of superoxide dismutase (P0.05) increased significantly. The inhibition of EsGSK3 activity after 6 hours, the expression level of Chinese mitten crab blood lymphocyte EsI kappa B mRNA significantly from Eriocheir sinensis cDNA library identified protein kinase B (Akt) gene, and named it the EsAkt. ORF 1395 BP, encoding a protein with 464 amino acids. The theoretical molecular weight of 53.17 kDa, the isoelectric point of 5.83. based on RT-PCR analysis, in all tissues examined, including blood cells, hepatopancreas, gill, gonad, muscle and eyestalk were detected EsERK gene expression of mRNA, one of the highest in the the expression level in muscle. By Aeromonas hydrophila (A.hydrophila) 12 hours after stimulation, the expression level of Eriocheir sinensis EsAkt lymphocytes in mRNA were significantly increased (P0.05). These results indicate that EsGSK3 can be induced by TNF- alpha, inhibiting blood cell endocytosis Apoptosis,.EsERK inherent immune response activity and NO synthesis regulation and regulate immune related enzymes of the Chinese mitten crab can participate in the immune response by regulating expression of I kappa B alpha and TNF- transcription factor, inhibiting their function will affect the inflammatory reaction caused by PI3K/Akt and Ras/ERK EsAkt also pathway. Show a response to bacteria stimulation. This is the first study to show the GSK3, ERK and Akt as a key regulator of glycogen metabolism pathways, plays a role in regulating innate immune response of Chinese mitten crab, provide the basis for further research on the role of PI3K and ERK signal pathway in the prevention and treatment of infection and inflammation control.

【学位授予单位】：中国科学院大学(中国科学院海洋研究所)
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S945

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