虾蟹类甲壳动物糖代谢激素的免疫调控作用

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	虾蟹类甲壳动物糖代谢激素的免疫调控作用
	王琳
学位类型	硕士
导师	宋林生
	2017-05-10
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	海洋生物学
关键词	甲壳动物糖代谢激素高血糖激素胰岛素样肽免疫调节
摘要	葡萄糖作为机体主要的能源物质，在机体免疫调控过程中，能够通过改变代谢途径进行能量的重新分配，优化免疫供能。在脊椎动物中的研究发现多种糖代谢激素既能调控葡萄糖代谢，又能作为免疫调节分子，直接参与免疫效应的调节，发挥了平衡机体内稳态的作用，但目前在无脊椎动物，关于糖代谢激素免疫调节作用的研究十分有限。本研究以甲壳动物凡纳滨对虾（Litopenaes vannamei）和中华绒螯蟹（Eriocheir sinensis）为实验对象，利用分子生物学、细胞生物学等生物学技术手段，对甲壳动物凡纳滨对虾中两种高血糖激素LvCHH I和LvCHH II以及中华绒螯蟹胰岛素样肽（EsILP）的功能及免疫调控作用进行了初步研究。本研究克隆了凡纳滨对虾CHH家族中的两个成员（ADL27417和AAN86057），并将其重新命名为 LvCHH I 和 LvCHH II，发现其分别含有一个/两个甲壳动物内分泌激素结构域。LvCHH I 和 LvCHH II蛋白在凡纳滨对虾眼柄中具有相似的表达模式，主要分布在窦腺和视上神经节内分泌细胞中。白斑综合症病毒（WSSV）感染后，凡纳滨对虾眼柄中LvCHH I 和 LvCHH II在转录水平显著上升，同时血淋巴上清中葡萄糖含量在WSSV刺激后的12、24小时显著增加，而血淋巴细胞内葡萄糖含量在WSSV刺激后6小时显著下降，12小时后再次上升。将LvCHH I的重组蛋白注射到凡纳滨对虾体内，发现血淋巴葡萄糖水平在刺激后0.5小时开始增加，并在3小时达到最大值，而LvCHH II重组蛋白刺激的凡纳滨对虾血淋巴葡萄糖在开始的0.5小时迅速下降，随后在3小时显著上升。血淋巴细胞中活性氧(ROS)水平在LvCHH I重组蛋白刺激后3-6小时显著降低，然而LvCHH II重组蛋白处理后未见ROS水平的显著变化。LvCHH I 和LvCHH II均能够定位于凡纳滨对虾的血淋巴细胞膜，并诱导原代培养的血淋巴细胞内抗病毒相关的免疫效应分子表达水平的变化：rLvCHH I孵育3小时后的血淋巴细胞中超氧化物歧化酶和LvRelish在转录水平的表达量均显著上升，抗菌肽分子crustin 和 penaeidin-4在转录水平的表达量也显著增加。与rLvCHH I不同，与rLvCHH II孵育后的原代血淋巴细胞中，超氧化物歧化酶和LvRelish 转录水平的表达量未发生明显变化，但抗菌肽crustin 和 penaeidin-4 转录水平的表达量显著增加。克隆获得中华绒螯蟹EsILP基因，其开放阅读框为216 bp，编码71个氨基酸。其多肽序列中包含一个胰岛素家族中保守的胰岛素样生长因子结构域，在该结构域中含有4个高度保守的半胱氨酸位点。EsILP 的转录本及蛋白主要表达于中华绒螯蟹的肝胰腺和眼柄神经节及胸腹神经团中。注射EsILP重组蛋白后，中华绒螯蟹血淋巴上清中葡萄糖水平显著下降。嗜水气单胞菌刺激后，EsILP的转录本在代谢和内分泌组织中呈现出相反的响应模式：在肝胰腺中，嗜水气单胞菌刺激诱导EsILP 转录水平表达量的显著下降，而在血淋巴细胞和眼柄中，EsILP 转录本的表达水平在菌刺激后显著上升。上述研究结果表明，凡纳滨对虾LvCHH I属于CHH家族的I型多肽，它参与上调血清中的葡萄糖水平并可能参与血淋巴细胞内的过氧化物的清除，LvCHH II属于CHH家族的II型多肽，不参与调节血糖水平的持续上升。两种LvCHHs都能够响应WSSV刺激，并通过与血淋巴细胞结合，激活下游NF-κB信号通路，诱导多种免疫效应分子的表达，参与调控抗病毒免疫反应。在中华绒螯蟹中发现的EsILP分子具有胰岛素家族保守的氨基酸序列特征和降血糖活性，其转录本与蛋白在代谢和内分泌相关组织呈较高表达。在嗜水气单胞菌刺激后，EsILP在肝胰腺和血淋巴细胞中的相反表达模式暗示了该分子可能调控了两种组织中葡萄糖代谢的转变。在甲壳动物中，CHH和ILP能够响应病原刺激，参与调控免疫防御过程中的能量供给，其中CHH能够诱导免疫效应基因的表达，参与免疫反应的调控。
其他摘要	A robust immune response against invading pathogens is crucial for host to survive, which depends greatly on the well balance of metabolism. Increasing evidence has indicated that some glucose metabolic hormones, such as insulin, could modulate immune responses directly. In the present study, the dynamic changes of crustacean hyperglycemic hormone (CHH) in Litopenaeus vannamei and insulin-like peptide (ILP) in Eriocheir sinensis were inestigateed after immune stimulation, the distribution and metabolic role were also identified, and the immunomodulatory role of CHH was further explored in shrimp L. vannamei for better understanding of the immunomodulation of glucose metabolic hormone in invertebrate. CHH family is a group of ecdysozoans-specific peptide hormone involved in glucose metabolism and other biological events. In the present study, two members of CHH family (designated as LvCHH I and LvCHH II) in shrimp Litopenaeus vannamei with one and two crustacean neurohormone domains respectively were chosen to investigate their putative modulatory roles in both glucose metabolism and immune response. LvCHH I and LvCHH II were both expressed in the sinus gland and lamina ganglionalis of eyestalks and were significantly induced after white spot syndrome virus (WSSV) infection. Meanwhile, significant increases of hemolymph glucose levels were observed in shrimp at 12 and 24 h after WSSV infection while the glucose inside the hemocytes decreased at 6 h and then increased at 12 h. Gain-of-function of rLvCHHs was subsequently conducted in vivo by injecting the recombinant proteins (rLvCHH I and rLvCHH II). The hemolymph glucose increased significantly from 0.5 h to 3 h after the shrimps received an injection of rLvCHH I, while it decreased at 0.5 h and increased afterward at 3 h post rLvCHH II injection. At the meantime, significant decreases of reactive oxygen species level in hemocytes were observed at 3 h and 6 h post rLvCHH I injection, while it remained unchanged in rLvCHH II injection group. rLvCHH I and rLvCHH II could bind to the cytomembrane of primary shrimp hemocytes in vitro, and the expressions of superoxide dismutase and LvRelish increased when the hemocytes were incubated with rLvCHH I for 3 hours. Meanwhile, the expression of antimicrobial peptides, crustin and penaeidin-4, were also induced by rLvCHH I and rLvCHH II. These results demonstrated that host immune response, in addition to glucose metabolism, could be directly modulated by LvCHH family. The full-length cDNA of an ILP (designated EsILP) was identified by the conserved insulin-like growth factor domain (IIGF) from the genome information of Eriocheir sinensis. The 216 bp open reading frame encoded a putative protein of 71 amino acids. Multiple sequence alignment presented four conserved cysteine residues which were critical for the fundamental structure and function of IIGF domain. The EsILP transcripts could be detected in the hepatopancreas, eyestalk, muscle, gill, hemocyte, hemopoietic tissue, stomach, while the EsILP protein was detected in hepatopancreas and thoracic ganglion mass. Gain-of-function of EsILP was subsequently conducted in vivo by injecting the recombinant proteins. The hemolymph glucose decreased significantly from 6 h to 12 h, indicating its functional similarity with insulin. Besides, the temporal expression of EsILP mRNA in different tissue after Aeromonas hydrophila challenge was measured by real-time RT-PCR. It was shown that the EsILP mRNA expression decreased significantly in hepatopancreas while highly increased in eyestalk and hemocytes. In conclusion, the above results demonstrated the direct immunomodulation role of glucose-regulating hormone in crustaceans and would shed new light into understanding the interaction between glucose-regulating hormone and immune response in crustaceans.
学科领域	生物学 ; 分子生物学
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136583
专题	实验海洋生物学重点实验室
作者单位	中国科学院海洋研究所
第一作者单位	中国科学院海洋研究所
推荐引用方式 GB/T 7714	王琳. 虾蟹类甲壳动物糖代谢激素的免疫调控作用[D]. 北京. 中国科学院大学,2017.

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