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糖原代谢相关基因在中华绒螯蟹免疫应答中的调控作用
其他题名The regulation function of key molecules in glycogen metabolism in immune response of Chinese mitten crab Eriocheir sinensis
李晓炜
学位类型硕士
导师王玲玲
2017-06
学位授予单位中国科学院大学
学位授予地点青岛
学位专业海洋无脊椎动物免疫学
关键词中华绒螯蟹 糖原合酶激酶3 细胞外信号调节激酶2 蛋白激酶b 固有免疫
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(p<0.05),在48小时后逐渐降至初始水平(p>0.05)。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(p<0.05),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(p<0.05),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(p<0.05),在48小时逐渐降低(p<0.05)。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(p<0.05),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(p<0.05), 超氧化物歧化酶活性显著升高(p<0.05)。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(p<0.05)。
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。

其他摘要The immune system continues to perceive and respond to the threat of the environment which needs to consume a lot of energy, while lack of energy will lead to immune system suppression and reduced resistance, so the regulation of energy during the immune response is critical.Because immune cells lack the storage of nutrients such as glycogen, only the increased intake of nutrients from the microenvironment can sustain these reactions. In this study, Chinese mitten crab Eriocheir sinensis was used as a research object to study the effects of glycogen synthase kinase 3 and its upstream regulatory kinase, protein kinase B (Akt) and extracellular signal regulating kinase 2 (ERK2), to explore the role of PI3K and ERK signaling in the innate immune response of E. sinensis.
Glycogen synthase kinase-3 (GSK3) is a protein kinase that catalyzes serine / threonine, which was first identified as a modulator of glycogen synthesis. Recently, it has been considered to be the key regulator of the immune reaction. In the current research, a GSK3 homologous gene (designated as EsGSK3) was cloned from Chinese mitten crab, E. sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-β from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas, and EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p < 0.05), and then gradually decreased to the initial level at 48 h in comparison with control group (p>0.05). The mRNA expression of Lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and production of TNF-α was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. The catalase, alkaline phosphatase activity and nitric oxide (NO) synthesis levels were significantly enhanced.These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF-α production and inhibiting haemocyte phagocytosis.
ERK (extracellular signal-regulated kinases), the upstream kinase of GSK3 pathway, was considered as a regulator of the metabolism of a series of genes in MAPK signaling pathway. In the present study, a homolog gene of ERK (designated as EsERK) was cloned from Chinese mitten crab E. sinensis. The open reading frame (ORF) of EsERK was of 1746 bp, encoding a protein of 581 amino acids. The EsERK mRNA expression levels in haemocyte increased significantly at 6 h after A. hydrophila challenge (p < 0.05), and then gradually decreased till 48 h (p < 0.05). After injecting dsERK into E. sinensis for 24 h, the mRNA transcripts of IκB (Inhibitor of nuclear factor kappa-B) rised significantly (p< 0.05) and superoxide dismutase (SOD) activity was also significantly increased (p< 0.05). Moreover, after lithium was injected into crabs as the specific inhibitor of EsGSK3, the IκB mRNA expression was significantly up-regulated at 6 h post injection (p < 0.05).
A novel Akt gene was identified from the cDNA library of E. sinensis and named as EsAkt. Its open reading frame (ORF) was of1395 bp, encoding a protein of 464 amino acids. The theoretical molecular mass of EsAkt was 53.17 kDa and its theoretical isoelectric point was 5.83. Based on RT-PCR analysis, the mRNA transcripts of EsERK were found expressed in all examined tissues, including haemocytes, gonads, hepatopancreas, gills, muscles and eyestalks. The highest expression level was detected in the muscle. The level of transcription of EsAkt was significantly up-regulated after being A. hydrophila challenge.
The above results indicated that EsGSK3 could regulate the innate immune response of E. sinensis by inducing TNF-α, inhibiting the phagocytosis of haemocytes, regulating the activity of immune-related enzymes and the synthesis of NO. EsERK could be involved in immune responses by regulating the expression of IκB and TNF-α transcription factors, influencing the inflammatory reactions mediated by PI3K/Akt and Ras/ERK pathways. In addition, EsAkt also showed the response to the bacteria stimulation. In this study, GSK3, ERK and Akt were identified from E. sinensis, not only as critical regulatory molecules of glycogen metabolism, but also vital regulators in the innate immune response , which provide the basis for in-depth study of the PI3K and Erk signaling pathway for the control of infection and inflammation in E. sinensis.
学科领域地球科学 ; 海洋科学 ; 海洋生物学
语种中文
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/136677
专题实验海洋生物学重点实验室
作者单位1中国科学院海洋研究所,2中国科学院大学
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李晓炜. 糖原代谢相关基因在中华绒螯蟹免疫应答中的调控作用[D]. 青岛. 中国科学院大学,2017.
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