河蟹flotillin样蛋白在CHH神经多肽激素调控血糖代谢中的作用研究

Crustacean hyperglycemic hormone (CHH) and its structurally related peptides, molt-inhibiting hormone (MIH), gonad-inhibiting hormone (GIH), form a neuropeptide family in crustaceans called the CHH-family. Members in this family play a pivotal role in the regulation of many important biological events including carbohydrate metabolism, molting, and gonadal development. To date, the CHH-family members have only been discovered from crustaceans. Besides, the targets of these hormones and their function have not been clearly identified. Transcriptomics analysis of genes expression followed by artificial eyestalk ablation of the mitten crab was carried out by our team. Among many unigenes which show great changes in the expression level, we select one gene which expressed membrane protein closely related to CHH, flotillin. In vertebrate, this protein usually acts as a signal station which transfers the signal from the outside of the cell into the inside. This project will choose the Chinese mitten crab, which is an important economic crustacean, as our research object and parse the molecular structure of this flotillin-like protein. With further transcriptomics analysis, we will try to verify the the binding activity between CHH and flotillin. Besides, the impact on the downstream signaling pathway of flotillin will also be clarified by gene silencing technology. The expected results will clarify the key target molecules of CHH in its regulation of glucose metabolism. Moreover, they will provide a valuable reference for the future research on the signal transduction mechanism of the carbohydrate metabolism regulated by CHH and the manual control of both crab basal metabolism and growth.

甲壳动物眼柄CHH家族神经多肽激素包括高血糖激素（CHH）、蜕皮抑制激素（MIH）、性腺发育抑制激素（GIH）等，调控着血糖代谢、蜕皮生长和性腺发育等重要的生理功能。CHH家族神经多肽激素迄今仅在甲壳动物中被发现，且尚不清楚它们的作用靶标分子、调控通路和机制。为了筛选CHH作用的靶标分子，本实验室以重要经济甲壳动物河蟹为研究对象，以切除眼柄前后的河蟹肝胰腺为样品，进行了转录组学和表达谱的初步分析，发现flotillin 样膜蛋白在糖代谢通路中发挥信号接收和传递功能，很可能起到了靶标分子的作用。本研究拟利用组学分析进一步验证CHH与flotillin样蛋白的相互关系，利用基因沉默等技术研究flotillin样蛋白对糖代谢中胞内下游信号通路的影响。预期结果有望阐明河蟹眼柄CHH调控糖代谢的关键靶标分子，为研究甲壳动物眼柄神经多肽激素调控糖代谢的复杂网络和人工控制河蟹的代谢生长提供参考。

甲壳动物眼柄CHH家族神经多肽激素调控着血糖代谢、蜕皮生长和性腺发育等重要的生理功能。CHH家族神经多肽激素迄今仅在节肢动物中被发现，且尚不清楚它的作用靶标分子、调控通路和机制。为确定CHH作用的靶标分子，本实验室以河蟹为研究对象，利用组学分析首先筛选出了一个可能的CHH靶标分子CHHBP，主要研究内容为验证CHH与CHHBP的相互关系，利用基因沉默等技术研究CHHBP对糖代谢中胞内下游信号通路的影响。. 我们首先利用生物大分子互作仪，证实CHHBP与CHH能够在体外产生互作。CHHBP主要表达在肝脏并定位于肝脏细胞的细胞膜上。实时定量PCR分析显示CHHBP的表达量在去眼柄后会出现显著的增加。对CHHBP的成功RNA干扰能使血糖水平降低，并且降低程度与去眼柄组中观察到的降血糖水平一致，表明CHHBP参与到了CHH调节的糖代谢通路中。此外，体外转染实验中，CHH诱导2-NBDG转运到细胞外的过程必须由CHHBP提供不可或缺的辅助。总之，这些发现暗示CHHBP可能作为受体参与到CHH调控的糖代谢信号通路中。由于CHH家族神经多肽激素调控糖代谢的通路和机制不同于其它生物，对这方面的研究还处于起步阶段；目前MIH及GIH调控作用的靶标分子经研究已基本确定为G蛋白偶联受体，但对CHH调控糖代谢的靶标及通路研究还是空白，因此我们的研究成果非常具有创新性以及重要的理论和应用价值，为深入了解甲壳动物眼柄神经多肽激素调控糖代谢的复杂网络和人工控制河蟹的代谢生长提供参考。