刺参5-HT4R活性特征及生理功能初探

发布时间：2018-01-01 00:11

本文关键词：刺参5-HT4R活性特征及生理功能初探　出处：《浙江海洋大学》2017年硕士论文　论文类型：学位论文

【摘要】：刺参又称作仿刺参(Apostichopus japonicus),属于海参纲(Holothuroidea)刺参科(Stichopodidae),是我国重要的海洋经济物种。自上世纪80年代起,人工养殖就已经取得突破。但在养殖过程中,夏眠导致刺参体重下降的问题严重影响其养殖产量,揭示刺参夏眠调控过程及其机制是解决这一问题的关键。血清素是重要的单胺类神经激素,血清素受体(serotonin receptor,5-HTR)是典型的G蛋白偶联受体,在动物的摄食调控中发挥着重要作用。本文开展刺参血清素受体Aj5-HTR基因克隆和序列特征分析、亚细胞定位和受体类型鉴定、在夏眠周期中变动规律及在代谢调控中的功能等方面的研究,初步探讨了Aj5-HTR在刺参摄食代谢中的调控作用和机制。主要取得的研究结果如下:采用RACE克隆技术获得了Aj5-HT4R的全长序列,并对其基因序列特征进行了分析,结果表明,Aj5-HT4R cDNA序列(KX583229)全长1,544 bp,包含233 bp的5'UTR,1,011 bp的ORF和300 bp的3'UTR,可编码336个氨基酸。和其他G蛋白偶联受体的结构一样,Aj5-HTR具有典型的七次跨膜结构。并利用系统进化分析发现,血清素受体明显分为六支:5-HTR1亚型,5-HTR2亚型,5-HTR4亚型,5-HTR5亚型,5-HTR6亚型,及5-HTR7亚型受体。Aj5-HT4R和5-HTR4聚为一支,与棘皮类动物紫海胆5-HTR4-like相似性为40%。通过荧光定量PCR基因表达定量检测分析了Aj5-HT4R在不同夏眠时期刺参组织中的表达特征。结果表明,Aj5-HT4R基因在刺参不同组织器官中广泛表达。刺参夏眠期间Aj5-HT4R在消化道、肌肉及呼吸树等组织中基因表达量呈现不同程度的下降,其中呼吸树中下降极显著。利用刺参活体生理实验,初步检测了5-HT作用下刺参呼吸树、消化道、肌肉中糖代谢相关的磷酸果糖激酶AjPFK的表达量。结果显示,相对于对照组(注射磷酸缓冲液),5-HT注射组在肌肉组织中的AjPFK表达量有显著升高(P0.05)。结合上述研究结果,说明5-HT及其受体5-HTR与刺参夏眠代谢调控存在关系。利用细胞生物学研究方法,对Aj5-HT4R受体性质及亚细胞定位进行分析。利用构建好的Aj5-HT4R-EGFP表达载体,转染至HEK293细胞中,用荧光染料DiI染膜、DAPI染核,在激光共聚焦显微镜下观察发现Aj5-HT4R主要定位在细胞膜上,与Aj5-HT4R序列中存在七次跨膜结构的预测结果相一致,表明Aj5-HT4R是一个膜受体,为下一步研究验证其功能奠定了基础。另一方面,通过一定浓度梯度的5-HT刺激转染有Aj5-HT4R-EGFP表达载体的HEK293细胞,结果显示Aj5-HTR在一定浓度(0、100pM、10nM、1μM)配体刺激下出现内吞。为深入揭示Aj5-HT4R在刺参夏眠调控中的功能及其作用机制,我们运用细胞信号转导研究平台,将构建的Aj5-HT4R-Flag转染到HEK293细胞中,检测5-HT作用下Aj5-HT4R介导的胞内cAMP浓度积累特征,初步分析其信号转导特征。以上结果显示,本论文中获得的刺参受体蛋白为5-HT4R并具有介导胞内cAMP信号的活性。本论文的研究结果对于今后进一步阐明刺参夏眠代谢调控机制,指导5-HT在棘皮动物等低等动物中的功能及生理调控作用机理相关研究。
[Abstract]:Sea cucumber Apostichopus japonicus (also known as Apostichopus japonicus), which belongs to the Holothuroidea (Holothuroidea) morinaceae (Stichopodidae), is China's economically important marine species. Since the last century since 80s, artificial breeding is a breakthrough has been made. But in the breeding process, resulting in the decrease of body weight of Apostichopus japonicus aestivation affect the aquaculture production seriously, revealing aestivation regulation process and its mechanism is the key to solve this problem. Serotonin is an important monoamine neurohormone, serotonin receptor (serotonin receptor 5-HTR) is a typical G protein coupled receptors, play an important role in the regulation of food intake in animal. Analysis of sea cucumber serotonin receptor Aj5-HTR gene cloning and sequence characteristics in subcellular localization and receptor type identification, change of law and in the regulation of metabolism function in the aspects of aestivation period, preliminary study of Aj5-HTR in sea cucumber The regulation effect and mechanism of feeding metabolism. The major findings are as follows: the full-length sequence of Aj5-HT4R cloned by RACE technology, and the gene sequences were analyzed, the results show that the Aj5-HT4R cDNA sequence (KX583229) was 1544 BP, containing 233 BP 5'UTR, 1011 BP ORF and 300 BP 3'UTR, encoding 336 amino acids. The same structure and other G protein coupled receptors, Aj5-HTR has seven transmembrane structure typical. And the use of phylogenetic analysis found that serotonin receptors were divided into six branches: 5-HTR1 subtype, 5-HTR2 subtype, 5-HTR4 subtype, 5-HTR5 subtype, 5-HTR6 subtype, and 5-HTR7 receptor subtype.Aj5-HT4R and 5-HTR4 were clustered into one clade, and echinoderms animal Anthocidaris 5-HTR4-like similarity to 40%. PCR gene by fluorescent quantitative quantitative analysis the expression characteristics of Aj5-HT4R in different period of sea cucumber aestivation expression. The result showed that the Aj5-HT4R gene is widely expressed in different tissues and organs. During aestivation of sea cucumber Aj5-HT4R gene expression in the digestive tract, respiratory muscle and tree in tissues showed different degrees of decline, which decreased significantly in the respiratory tree. The use of sea cucumber in vivo physiological experiment, preliminary detection of sea cucumber respiratory tree, under the action of 5-HT digestion that expression of sugar metabolism related muscle phosphofructokinase AjPFK. The results showed that, compared with the control group (injection of phosphate buffer), 5-HT injection group in the muscle tissue of AjPFK expression increased significantly (P0.05). Based on the above results, indicating the existence of 5-HT and its receptor 5-HTR and aestivation of metabolic regulation. Using the methods of cell biology, Aj5-HT4R receptor properties and subcellular localization were analyzed. Using the constructed Aj5-HT4R-EGFP expression vector was transfected into HEK293 cells with fluorescent dye. DiI dye film, DAPI staining, observed that Aj5-HT4R mainly localized in the cell membrane under confocal laser scanning microscope, and Aj5-HT4R sequence in the seven transmembrane structure prediction results are consistent, indicating that Aj5-HT4R is a membrane receptor, which laid the foundation for the next research verify its function. On the other hand, by a certain concentration gradient of 5-HT stimulated Aj5-HT4R-EGFP expression vector transfected HEK293 cells. The results showed that Aj5-HTR in certain concentration (0100pM, 10nM, 1 M) endocytosis of ligand stimulation. There is to reveal Aj5-HT4R in aestivation in the regulation of function and mechanism, we use cellular signal transduction research platform, will the construction of Aj5-HT4R-Flag transfected into HEK293 cells, the accumulation characteristics of cAMP concentration detection under 5-HT Aj5-HT4R mediated intracellular signal transduction, a preliminary analysis of its characteristics. The above results obtained in this thesis show that sea cucumber 5-HT4R receptor protein and is mediated by intracellular cAMP signaling activity. The results of this study to further clarify the regulatory mechanism of aestivation of metabolism, function and physiological regulation mechanism in animal Echinodermata animal guidance in low 5-HT.

【学位授予单位】：浙江海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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