虹鳟的骨代谢及其调控机理

Bone serves as an important storage point for calcium, as it contains 99% of the total body calcium. Mineralization and mobilisation of calcium respectively represents the bone formation and bone resorption process, which are the two important aspects of bone metabolism. Accordingly, calcium metabolism may reflect bone metabolic homeostasis as it refers to all the movements of calcium into and out of body compartments. As an important component of the salt metabolism, calcium metabolism in fish varies with changing environmental salinity. Understanding the influence of changes in environmental salinity on calcium metabolism would be the first step to elucidate the response of teleost bone metabolism to environment change. Similar to other vertebrates, teleost skeleton contains bone-forming cells (osteoblasts) and resorptive cells (osteoclasts). Examining their activity variation as well as the gene regulatory work would provide new insights into regulatory mechanism of bone metabolism. In this study, morphologic, physiologic and cytologic research will be performed to investigate the effects of external salinity on calcium metabolism and bone metabolism in rainbow trout. Additionally, osteoblasts and osteoclasts will be respectively collected and used for transcriptome analysis to clarify the regulatory mechanism of bone metabolism in response to altered salinity. Relative results may help understanding teleost bone metabolism and its regulatory mechanism at the cellular level, and lay a theoretic foundation for development of the trout aquaculture industry.

硬骨鱼类骨骼是鱼体的钙库，其体内约99%的钙分布于骨组织。骨的新陈代谢包括骨的形成和吸收，由成骨、破骨细胞行使功能，主要表现为钙的生物矿化及再活化，其动态过程则由钙代谢所表征。钙代谢指钙在体内的吸收、分布及排泄，是无机盐代谢的重要组成。鱼类的无机盐代谢等生理活动与水环境离子强度（盐度）密切相关，研究盐度与钙代谢的关系是阐明鱼类骨代谢调控机理的切入点；同时，研究成骨、破骨细胞功能的动态变化及其调节机制是认识骨代谢的关键。本项申请拟以虹鳟为研究载体，紧扣“盐度变化—无机盐代谢（钙代谢）—骨代谢”这一主线，分别从形态、生理及细胞水平考察盐度变化对虹鳟钙代谢、骨代谢的影响，明确无机盐代谢及骨代谢之间的内在联系；对成骨、破骨细胞进行转录组测序，从分子水平探讨骨代谢应对盐度变化的调节机制，为探明硬骨鱼类骨代谢的调控机理及其应对环境变化的演化规律提供新视角，为虹鳟养殖生产实践提供参考。

硬骨鱼类骨骼的新陈代谢主要表现为钙的生物矿化及再活化，这一无机盐代谢活动与水环境离子强度（盐度）密切相关。本研究以“盐度对鱼类钙代谢的影响”作为阐明鱼类骨代谢调控机理的切入点，选取虹鳟作为研究载体，分别从形态、组织、细胞、生理及分子等不同水平考察了盐度变化对虹鳟骨代谢的影响。首先，利用硬骨-软骨双染色技术及组织切片等方法对骨骼发育过程进行系统描述，利用X光扫描成鱼全骨骼，利用ICP-MS、micro CT等技术检测不同发育期骨组织的显微结构及钙、鳞元素组成情况，摸清了虹鳟早期骨骼形骨骼发生、发育过程及骨组织形态、元素组成等特征。随后，对虹鳟幼鱼开展为期21天的盐度驯化实验。利用电感耦合等离子体质谱（ICP-MS）检测外骨骼（鳞）中的钙、磷元素含量；利用von Kossa染色法对磷酸钙进行染色，分析鳞组织中钙的含量及分布特征；采用酶组织化学方法分别对成骨细胞、破骨细胞活性标记--ALP、TRAcP活性进行检测；采用Illumina Hiseq 4000平台进行全转录组测序，分析骨组织中mRNA及ncRNA（miRNA、lncRNA等）表达水平的变化趋势，筛选出了数个可能参与虹鳟钙代谢、骨代谢的关键基因。此外，通过对盐度驯化后的虹鳟幼鱼进行刮鳞处理使鳞再生，利用组织切片、ICP-MS等技术分别检测了鳞再生过程中新鳞的组织形态及元素含量，采用全转录组测序等技术分析了盐度驯化影响鳞再生的分子机制。最后，通过对盐度驯化后的虹鳟幼鱼腹腔注射钙调节因子（鲑降钙素或钙调蛋白抑制剂），基于鳞组织中钙、磷元素含量变化情况，以及mRNA和ncRNA的表达水平的变化趋势，探讨了“盐度驯化—钙代谢—骨代谢”这一主线的虹鳟钙代谢调节途径，明确了钙代谢与骨代谢之间的内在联系。本研究获得的结果可为探明硬骨鱼类骨代谢的调控机理及其应对环境变化的演化规律提供新视角，为虹鳟养殖生产实践提供参考。