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题名: 长牡蛎神经内分泌免疫系统调节机制的初步研究
作者: 刘兆群1,2
学位类别: 博士
答辩日期: 2017-05-10
授予单位: 中国科学院大学
授予地点: 北京
导师: 宋林生
关键词: 长牡蛎 ; 神经内分泌 ; 免疫调节 ; 细胞因子 ; 血淋巴细胞
学科分类: 生物学 ; 水产学
学位专业: 海洋生物学
中文摘要:     神经内分泌免疫调节系统(neuroendocrine-immune regulatory system,简称NIE系统)是指由神经系统、内分泌系统和免疫系统组成的互惠统一的整体。三个系统之间通过释放神经递质、神经调质、激素和细胞因子等信号分子进行双向调节,共同维持机体的内稳态。目前,关于高等动物NEI系统的研究比较透彻,研究热点主要集中在神经性精神紊乱、生长发育调控、疾病的发病机理等方面。近年来,无脊椎动物,特别是软体动物NEI系统的研究取得了重大进步,已逐步由简单的形态学观察和分子鉴定发展到调控机制和生理意义的探究中。     本文从软体动物几种关键神经递质及其受体入手,利用组学、生物信息学、细胞生物学和免疫学等手段,较深入地揭示了长牡蛎NEI系统的结构组成、激活机制和免疫调控模式。
利用神经显微解剖、石蜡组织切片和HE染色等方法,初步探究了长牡蛎神经系统的结构组成及形态特征。长牡蛎的神经系统主要由1对头神经节、1个脏神经节和与神经节相连的神经索及神经纤维组成。头神经节位于唇瓣基部,左、右各1个;脏神经节左右合并为一个半圆形淡黄色组织,位于闭壳肌前端平滑肌与横纹肌之间的凹陷内。由脏神经节伸出两条主神经索,沿闭壳肌向前经过血窦和肝胰腺,最终与头神经节相连接,构成完整的长牡蛎神经系统。在对长牡蛎神经节及神经元胞体进行观察后发现,头神经节为椭圆形结构,外周被结缔组织、肌肉组织和少量脂肪组织所包裹。在神经节内部,神经元细胞聚集成团,神经纤维则汇集成束。头神经节通过节间神经索与内脏神经节相连接,并由节内神经细胞发出神经纤维分布于唇瓣之中。较之头神经节,脏神经节体积更大,神经纤维更长,所占神经节体积比也更大。
    从长牡蛎中分别克隆得到了1个脑啡肽(ENK)受体基因CgDOR,1个乙酰胆碱(ACh)受体基因CgmAChR-1和1个去甲肾上腺素(NE)受体基因CgA1AR-1CgDOR全长1104 bp,编码一段长度为367个氨基酸的肽段;在Cys95, Cys125 和 Cys174处发现3个保守的半胱氨酸位点,其中PROSITE 预测出在Cys95 和 Cys174间可以形成一个二硫键。CgmAChR-1全长1983 bp,编码一段长度为660个氨基酸的肽段;CgmAChR-1是一类毒蕈碱型乙酰胆碱受体,并且有可能属于m5型mAChR。CgA1AR-1全长1149 bp,编码一段长度为382个氨基酸的肽段;CgA1AR-1首先与Homo sapiensBos taurus的A1ARs聚为一支,再与Homo sapiensRattus norvegicus等动物的A2ARs聚为一簇,最后再与其它动物的BARs相聚类。上述三种受体基因在血淋巴细胞、肝胰腺、肾脏、闭壳肌、外套膜、鳃和性腺中均呈组成型表达。
    长牡蛎神经内分泌系统可以被内外因子激活。其中,肿瘤坏死因子(CgTNF-1)能通过促分裂素原活化蛋白激酶(MAPK)信号转导通路激活转录因子NF-κB和热休克转录因子(HSF),从而调控血淋巴细胞的凋亡、氧化还原反应、神经性调控以及蛋白折叠过程。细菌刺激和热应激能够促使长牡蛎幼虫的儿茶酚胺系统被激活,合成并释放多巴胺、肾上腺素和去甲肾上腺素等神经递质,并进一步调控各项生理过程,最终使个体能够更好地生存。另外,脂多糖(LPS)刺激能够迅速激活成体长牡蛎的神经内分泌系统,促进神经递质的合成和释放。LPS刺激还能够直接激活长牡蛎血淋巴细胞的神经内分泌系统,促进其合成和释放神经递质。
    长牡蛎血淋巴细胞作为免疫细胞,通过控制其细胞膜表面神经递质受体的表达水平和结合活性,选择性介导神经性免疫调节。当牡蛎血淋巴细胞受到多种神经递质协同作用时,受体更倾向于结合NE这一类的神经递质,而非ENK。同时长牡蛎膜受体对配体的结合也具有时序性,在免疫应答早期和晚期,分别通过介导以ENK为主的免疫上调作用,及以NE为主的免疫下调作用,维护内环境的稳定。此外,长牡蛎血淋巴细胞也可以作为神经内分泌细胞,通过自分泌或旁分泌的方式调控免疫应答。LPS刺激能够诱导长牡蛎血淋巴细胞合成和分泌神经递质,血淋巴细胞源的神经递质ACh和NE能够显著下调细胞的免疫应答水平。同时,此种神经递质的调控作用也是经由细胞表面的受体介导的。当用膜受体抑制剂预先封闭受体后,血淋巴细胞自身分泌的神经递质对免疫过程的调控作用也被显著抑制了。
多种细胞因子、转录因子和信号转导通路参与了长牡蛎的神经内分泌免疫调控。神经递质与长牡蛎血淋巴细胞膜表面受体结合后,通过EκbB、p53和EGF等信号通路,调节细胞因子TNF和转录因子NF-κB、AP-1等分子的表达。同时,细胞蛋白复合体组装,细胞膜脂质代谢,细胞内稳态和细胞程序性死亡等过程,也极有可能在神经递质介导的免疫调控作用中发挥核心作用。最终,长牡蛎的细胞免疫和体液免疫过程通过上述途径被激活,从而实现神经性免疫调节,帮助机体清除病原,得以更好地生存。
    上述研究结果表明,长牡蛎神经内分泌系统既可以被外部环境因子激活,又能够被体内细胞因子激活。被激活的神经内分泌系统释放ACh、NE和ENK等神经递质,通过EκbB、p53和EGF等信号通路,调节细胞因子TNF-α和转录因子NF-κB、AP-1等分子的表达,最终影响长牡蛎的细胞免疫和体液免疫应答过程,实现神经性免疫调节。同时,长牡蛎血淋巴细胞既可以作为重要的免疫细胞,有选择性地主动介导神经递质的免疫调控作用;又可以作为一类新型的神经内分泌细胞,通过自分泌或旁分泌的方式在血淋巴细胞自身水平行使神经性免疫调节功能。以长牡蛎为代表的软体动物已经进化出了与高等动物类似的NEI系统,并在机体环境适应和稳态调节中发挥了关键作用。

关键词:长牡蛎,神经内分泌,免疫调节,细胞因子,血淋巴细胞
英文摘要:     The neuroendocrine-immune (NEI) regulatory network consists of nervous system, endocrine system and immune system, which carries a reciprocal regulation to maintain homeostasis in the host with the involvement of signaling molecules, such as neurotransmitters, hormones and cytokines. By far, most of the research conducted in vertebrates focus on neuropsychiatric disorders, developmental programming, disease pathogenesis, and comparative approaches, while little is known about the NEI network in invertebrates, especially in molluscs. In the present study, the structural basis, molecular composition, regulation patterns, as well as the physiological significances of the NEI network in molluscs have been explored by using multiple approaches including RNA-seq, bioinformatical analysis and biological validation.
    The nervous system of oyster is composed of two cerebral ganglia (CG) under the palps, one visceral ganglia (VG) next to the adductor muscle, and the nerve cords and nerve fibers which connect them together. One opioid receptor for enkephalin (ENK) (CgDOR), one muscarinic acetylcholine (ACh) receptor (CgmAChR-1) and one norepinephrine (NE) receptor (CgA1AR-1) were identified and cloned from oyster haemocytes. The ORF (open reading frame) of CgDOR was 1104 bp, encoding a protein of 367 amino acids. Three conserved cysteine residues (Cys95, Cys125 and Cys174) were identified by the alignment of CgDOR with other two vertebrate DORs, and the conserved Cys95 and Cys174 were predicted to form an important disulphide bridge by PROSITE. The ORF of CgmAChR-1 was 1983 bp and the polypeptide sequence contained 660 amino acids with a predicted molecular mass of 74.08 kDa. According to the phylogenic tree, CgmAChR-1 was closely matched with m5 muscarinic receptors found in invertebrates. Also, the nucleotide sequence of CgA1AR-1 was 1149 bp, and the polypeptide sequence contained 382 amino acids with the molecular mass predicted as 43.18 kDa. According to the phylogenic tree, CgA1AR-1 was closely matched with A1ARs previously identified in vertebrates. The mRNA transcripts of all these three receptors were detected in oyster tissues such as haemocytes, gonad, gill, mantle, adductor muscle, kidney and hepatopancreas.
    The neuroendocrine system of oyster could be activated by CgTNF-1 through the activation of MAPK signal pathway and the transcription factor NF-κB and HSF, regulating the apoptosis, redox reaction, neuro-regulation and protein folding in oyster haemocytes. Besides, acute heat and bacterial stress could significantly inhibit larval development and suppress immune response of oyster C. gigas larvae. And the neuroendocrine immunomodulation, especially the catecholaminergic regulation, played an indispensable role in the stress response of molluscan larvae. Moreover, the neuroendocrine system could also be triggered by LPS, resulting in the synthesis and release of neurotransmitters in both individual oyster and primarily cultured haemocytes.
    Oyster haemocytes were able to work as just immune cells and form the simple “nervous-haemocyte” neuroendocrine immunomodulatory axis-like pathway to mediate neuronal immunomodulation promptly by controlling the expression of specific neurotransmitter receptors on cell surface and modulating their binding sensitivities. Meanwhile, oyster haemocytes could also serve as a new source of cholinergic and adrenergic neurotransmitters to execute a negative regulation on innate immune response with similar autocrine/paracrine self-regulatory mechanisms identified in vertebrate lymphocytes.
    Moreover, oyster neurons might be activated after LPS or heat stress and then release neurotransmitters to modulate the immune response by promoting the expression of TNF and translocation of NF-κB in cytoplasm and nuclei of haemocytes. And, the immunomodulation of neurotransmitters ACh and ENK could induce differentially expression of lots of proteins and affect both humoral (SOD and CAT) and cellular (haemocyte apoptosis and phagocytosis) immunity through p53 signaling pathway, EGF-R-ErbB signaling pathway, and FcγR signaling pathway.
    All these results suggested that an analogous NEI network was also exist in molluscs, which was simple-in-structure but complex-in-function. This network could be activated by both cytokines and environmental stressors. The activated neuroendocrine system then release neurotransmitters, such as ACh, NE and ENK to modulate cellular and humoral immune responses through the EkbB, p53 and EGF signaling pathways, regulating the expression of cytokines and transcription factors including TNF-α, NF-κB and AP-1. In particular, oyster haemocytes could work as immune cells by mediating the immunomodulation of neurotransmitters released from the neuroendocrine system, and as a new sort of neuroendocrine cells by producing neurotransmitters and preforming autocrine/paracrine self-regulatory processes. Results in the present study would contribute to a better understanding of the NEI network lower forms of live, and shed light on the study of the evolution of NEI network.
 
Key Words: Crassostrea gigas, Neuroendocrine, Immunomodulation, Cytokines, Haemocyte
 
语种: 中文
内容类型: 学位论文
URI标识: http://ir.qdio.ac.cn/handle/337002/136570
Appears in Collections:实验海洋生物学重点实验室_学位论文

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作者单位: 1.中国科学院海洋研究所
2.中国科学院大学

Recommended Citation:
刘兆群. 长牡蛎神经内分泌免疫系统调节机制的初步研究[D]. 北京. 中国科学院大学. 2017.
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