中华绒螯蟹肠道中组织胺和5-羟色胺分布和功能的研究

The Chinese mitten crab Eriocheir sinensis (commonly known as crab) is an indigenous and important economic crustacean in China. In recently years, Eriocheir sinensis aquaculture has grown rapidly in china.The protein -rich feeds are often utilized in Eriocheir sinensis aquaculture. However, stale feeds contain high level of biogenic amines, such as histamine（HA）and 5-hydroxytryptamine (5-HT). HA and 5-HT could significantly impact the growth of aquatic animals. However, to date, no published information is available in regarding to the mechanism of action for these amines. A number of techniques, such as immunohistochemistry, confocal, nerve tracing, HPLC and electrophysiology patch clamp technique etc, will be used in this study．In the present study, firstly, we will show that the histaminergic and serotonin neurons in gut of Eriocheir sinensis exhibit both HA-like and 5-HT-like immunoreactivity. The HA, 5-HT and synthesizing or metabolizing enzymes of HA and 5-HT in gut will be analyzed. The receptor types and the cell types of producing HA and 5-HT will be determined. Secondly, this study will understand the regulatory role of these amines in Eriocheir sinensis gut by observed the changes of gut contractile, intestinal epithelial proliferation, digestive enzymes and antimicrobial activity etc. Finally, we will clarify the mechanism of biogenic amines on the growth of aquatic animals based on observation of digestive physiology. The receptor antagonist types of HA and 5-HT will be preliminarily determined to relieve gastrointestinal dysfunction caused by HA and 5-HT. This study will provide large amounts of information about the gut neuromodulation of crustacean. This study will also be as a theoretical basis for the stale feeds (containing HA or 5-HT) scientific using.

中华绒螯蟹（俗称河蟹）是我国特有且重要的经济类甲壳动物。人工养殖量逐年增加。在实际饲喂中常有大量高蛋白质饵料使用。这些饵料腐败后会含有大量生物胺，如：组织胺（HA）和5-羟色胺（5-HT）。HA和5-HT会对水产动物生长产生影响，但机理不明。本研究将运用免疫组化、激光共聚集、神经示踪、高效液相色谱和电生理膜片钳等多项技术。首先，弄清河蟹肠道内HA和5-HT神经和递质分布，对相应递质及合成酶或代谢酶含量进行测定，确定其相应受体类型和产生此类物质的细胞类型。然后，从肠道收缩、肠上皮增殖、消化酶和抑菌能力变化等方面，弄清两种胺在河蟹肠道中的调节作用。最后，从消化生理角度阐明生物胺对水产动物生长影响的机理。初步确定能解除由它们引发的胃肠道功能异常的受体拮抗剂类型。本研究将丰富甲壳动物肠道神经调节资料。为变质（含有HA或5-HT）饲料的科学使用提供理论依据。

中华绒螯蟹（俗称河蟹）是我国特有且重要的经济类甲壳动物。在实际饲喂中常有大量高蛋白质饵料使用。这些饵料腐败后会含有大量生物胺，如：组织胺（HA）和5-羟色胺（5-HT）。HA和5-HT会对水产动物生长产生影响，但机理不明。本研究将运用免疫组化、高效液相色谱和分子生物学等多项技术。首先，弄清河蟹肠道组织学结构，并对主要上皮细胞类型进行鉴别。其次，弄清了河蟹中肠和后肠中5-羟色胺合成酶和5-羟色胺受体2(5-HTR2)的分布情况，已成功克隆到5-HT受体7全长，获得5-HT受体1、受体2和多巴胺等多种生物胺基因的核心片段。发现5-HT受体2有促进肠道运动的作用，其激动剂α-Me-HT有进一步促进作用。接着，利用肠道铺片、特殊染色和电镜观察等手段发现了河蟹肠道收缩相关神经元和间质样细胞的分布，并分析了肠道收缩相关递质的水平。此外，对河蟹参与消化和免疫调节的血细胞和肝胰腺细胞进行了体外培养和功能分析。河蟹血细胞离体培养的最佳条件为：3×L-15培养基调整渗透压至1100 mOsM kg-1，pH 6.8，28℃。血细胞存活率5d可达＞60%。大颗粒血细胞胞质内有酸性和碱性磷酸酶、β-葡萄糖醛酸酶和酚氧化酶等活性物质。肝胰腺细胞体外培养72h存活率可达＞50%。然后，本研究发现饲料中组织胺的添加对河蟹消化结构和功能有不良影响。证明在高温收获季节死亡24h后的河蟹三种可食组织（肌肉、肝胰腺和性腺）不能被食用，这些组织内主要的生物胺类型是腐胺而不是组织胺。此外，尸胺、腐胺、酪胺和组胺可作为两种常见淡水鱼（餐条和红鳍鲌）新鲜度的评价指标。最后，本研究发现5-HT的前体氨基酸即色氨酸和5-HT下游的功能递质即褪黑素均在一定程度上参于了河蟹生长、运动、免疫和血糖水产的调节。综上，这些结果不但有助于弄清河蟹肠道消化生理和免疫调节机制，还有助于生产中变质（含有HA或5-HT）饲料的科学使用、饲料中相应氨基酸的添加和食品中生物胺的安全监控。