5-羟色胺能系统在长牡蛎应对干露胁迫中的调控机制研究

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	5-羟色胺能系统在长牡蛎应对干露胁迫中的调控机制研究
	董文静
学位类型	硕士
导师	王玲玲
	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	长牡蛎 5-ht 5-ht受体干露胁迫抗氧化作用 Crassostrea Gigas 5-ht 5-htr Air Exposure Anti-oxidation
其他摘要	5-羟色胺（5-HT）能系统作为神经内分泌系统的重要组成部分，在脊椎动物和无脊椎动物的许多生理过程中发挥着重要作用。它不仅调节机体的免疫应答、能量代谢、情绪调控、生长繁殖等过程，还参与了机体在环境压力下的应激反应。当压力来临时，机体的5-HT 神经内分泌系统被激活并释放5-HT，5-HT 与膜上的5-HT 受体（5-HTR）结合后共同发挥调控作用。目前软体动物5-HT 神经内分泌系统的组成特征，及其在应对环境胁迫时所发挥作用的相关报道还十分有限。本研究以长牡蛎为研究对象，利用免疫学、分子生物学和细胞生物学等手段，查明了软体动物5-HT 神经内分泌系统的分子组成及表达特征，明确了5-HT 神经内分泌系统对干露胁迫的响应模式，初步阐明了其在长牡蛎应对干露胁迫时发挥的调控作用。从长牡蛎中克隆获得了4 个5-羟色胺受体基因：Cg5-HTR1、Cg5-HTR2、 Cg5-HTR3 和 Cg5-HTR4，均为含有7 次跨膜结构域的G 蛋白偶联受体。 Cg5-HTR1 的cDNA 全长为1443 bp，编码一条由 480 个氨基酸组成的多肽链； Cg5-HTR2 的cDNA 全长为1239 bp，编码一条由 412 个氨基酸组成的多肽链； Cg5-HTR3 的cDNA 全长为1254 bp，编码一条由 417 个氨基酸组成的多肽链； Cg5-HTR4 的cDNA 全长为1008 bp，编码一条由335 个氨基酸组成的多肽链。4 个Cg5-HTR 的mRNA 转录本均在肝胰腺、血细胞及性腺中呈组成型表达，其中肝胰腺表达量最高。Cg5-HTR1 的mRNA 转录本在外套膜中也有分布； Cg5-HTR2 的mRNA 转录本在闭壳肌中有分布；Cg5-HTR3 和 Cg5-HTR4 的 mRNA 转录本分布较广，在鳃、闭壳肌、外套膜中均有分布。同时，5-HT 主要分布于长牡蛎外套膜边缘、肝小管间隙以及脏神经节，在鳃、唇瓣和性腺中也有少量零星分布，但未在闭壳肌中检测其存在。此外，在5-HT 刺激下，经Cg5-HTR3 重组质粒转染后的Hela 细胞内的二级信使Ca2+的水平显著提高，而用5-HTR 抑制剂Way-10063 或Metagoline 提前孵育转染质粒的Hela 细胞，其细胞内Ca2+水平未发生显著变化。干露胁迫后长牡蛎血清中5-HT 含量呈现显著上升趋势，在胁迫后期上升幅度尤为显著。长牡蛎外套膜中5-HT 表达水平在干露胁迫后同样呈现显著升高趋势。干露胁迫能够显著影响长牡蛎血细胞中Cg5-HTR 的 mRNA 表达水平，其中Cg5-HTR1 和Cg5-HTR3 的表达量在胁迫后期显著升高，Cg5-HTR2 的表达量在胁迫前期显著增加，而Cg5-HTR4 表达量呈现先上升后下降的趋势，在胁迫后期恢复初始水平。外源添加5-HT 能够显著抑制长牡蛎血细胞的凋亡，并显著提高血清中一氧化氮合酶（NOS）和超氧化物歧化酶（SOD）的活性。持续的干露胁迫能够提高长牡蛎血细胞的凋亡率、血清中SOD 的活性和过氧化氢（H2O2）的含量，并抑制血清中NOS 的活性和血细胞中CgTNF 的mRNA 表达水平。在干露胁迫过程中注射5-HT 后，长牡蛎血清中SOD 的活性显著升高，而H2O2 的含量显著降低；在胁迫应答前期血清中NOS 的活性显著上升，在胁迫应答后期血细胞的凋亡率显著下降，个体存活率显著提高。上述结果表明，长牡蛎体内存在相对完善的5-HT 神经内分泌系统，且5-HT 和Cg5-HTR 在各组织中广泛表达。干露胁迫下，5-HT 神经内分泌系统被激活， 5-HT 在血清和外套膜中的含量显著升高，Cg5-HTR 的mRNA 转录本在血细胞中的表达也发生显著变化。激活的5-HT 主要通过抑制细胞凋亡和增强抗氧化作用等机制来调控机体在干露胁迫下的应激反应，从而显著降低干露胁迫下长牡蛎的死亡率。 ; As an important component of the neuroendocrine system, serotonin (5-HT) system plays a major role in the regulation of various biological processes, including immune responses, energy metabolism, emotional behaviours, growth, reproduction, and stress responses to the changing environment, in both vertebrates and invertebrates. The 5-HT neuroendocrine system could be activated right after receiving an environmental stress or a stimulation, resulting in the synthesis and release of 5-HT to modulate the subsequent physiological processes responsive to these threats. By far, few researches have been investigated upon the molecular components of 5-HT neuroendocrine system, and the roles of 5-HT neuroendocrine system under air exposure in molluscs. In the present study, the molecular components and expression features of 5-HT neuroendocrine system, and the response pattern of 5-HT neuroendocrine system under air exposure were explored in oysters for better understanding of the modulation of 5-HT system under air exposure. Four 5-HT receptors (Cg5-HTR) including Cg5-HTR1, Cg5-HTR2, Cg5-HTR3 and Cg5-HTR4 were cloned from Pacific oyster Crassostrea gigas, with seven transmembrane regions. The cDNA sequence of Cg5-HTR1 was 1443 bp, encoding a polypeptide of 480 amio acids. The cDNA sequence of Cg5-HTR2 was 1239 bp, encoding a polypeptide of 412 amio acids. The cDNA sequence of Cg5-HTR3 was 1254 bp, encoding a polypeptide of 417 amio acids. The cDNA sequence of Cg5-HTR4 was 1008 bp, encoding a polypeptide of 335 amio acids. The mRNA transcripts of four Cg5-HTR were expressed in hepatopancreas, haemocytes and gonad, with the highest expression level detected in hepatopancreas. The mRNA transcript of Cg5-HTR1 was also expressed in mantle, while Cg5-HTR2 was expressed in adductor muscle. And, the mRNA transcripts of Cg5-HTR3 and Cg5-HTR4 were widely expressed and also determined in gill, adductor muscle and mantle. In addition, the tissue distribution of 5-HT was further investigated with immunoflouroscence. 5-HT was mainly distributed in visceral ganglion, the edge ofmantle and the gaps among hepatic tubules of hepatopancreas, but randomly and weakly expressed in gill, the exterior structure of labial palps and gonad. No positive signal was detected in adductor muscle. Furthermore, after recombinant plasmid of Cg5-HTR3 was transfected into Hela cells, the level of intracellular Ca2+ was significantly increased after the incubation of 5-HT. However, the effect could be inhibited by two different type antagonists (Way-10063 and Metagoline) of 5-HTR. 5-HT concentration increased significantly in haemolymph of the oysters after they were exposed to air, especially during the late stage of the exposure. Meanwhile, the change of 5-HT concentration in mantle was detected after air exposure. The positive signals of 5-HT were firstly appeared in the edge of mantle under normal condition, and the signals became stonger along with the duration of air exposure. Moreover, the mRNA expressions of the four Cg5-HTR were also increased significantly after air exposure. Specifically, the mRNA expressions of Cg5-HTR1 and Cg5-HTR3 in haemocytes were increased significantly at late stage, while Cg5-HTR2 mRNA was increased significantly at early stage. The mRNA expression of Cg5-HTR4 was increased during the early stage after treatment, then decreased and restored to the initial level during the late stage. The apoptosis rate of haemocytes was remarkably declined, while the activities of superoxide dismutase (SOD) and nitric oxide synthase (NOS) in haemolymph were significantly increased after an injection of 5-HT into oysters under air exposure. Continuous air exposure would increase the apoptosis rate of haemocytes, SOD activity and H2O2 level in haemolymph, but decrease the NOS activity and the mRNA expression of CgTNF of haemocytes in oysters. In addition, once the oysters received an extra injection of 5-HT during air exposure, the SOD activity in haemolymph was significantly elevated, and the H2O2 level in haemolymph was astonishingly reduced. Meanwhile, the NOS activity in haemolymph was significantly elevated during the early stage of air exposure, and the apoptosis rate of haemocytes was remarkably reduced, while the survival rate of oysters was significantly increased during the late stage. In summary, molluscs such as oysters C. gigas have evolved a relatively complete 5-HT neuroendocrine system, and its major molecular components including 5-HT and 5-HTR were widely distributed in all the important tissues. When the oysters were exposed to air, 5-HT neuroendocrine system could be activated and the levels of 5-HT in mantle and haemolymph, the mRNA expressions of the four Cg5-HTR were changed significantly after air exposure. The activated 5-HT could relieve the apoptosis and increase anti-oxidation ability of oysters, which played a key role in regulation of air exposure stress in oysters, and reduced mortality of oysters under air exposure.
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/337002/136552
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	董文静. 5-羟色胺能系统在长牡蛎应对干露胁迫中的调控机制研究[D]. 北京. 中国科学院大学,2017.

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