基于VA介导下RBP4对斜带石斑鱼肝脏脂肪代谢的调控机制

The fatty liver is one of the main factors limiting the healthy development of the seawater aquaculture, which might result from unbalanced nutritional diet. Prevention and control of fatty liver, which in order to through nutrition regulation measures of the aquatic animal has become a hot spot. The latest study in the model of mammals indicated that retinol binding protein 4 (RBP4) is a new type of fat source signal factor, which have important role in the regulation of fat metabolism. But, the synthesis and secretion of RBP4 is closely related to vitamin A (VA). However, about the relation among VA, RBP4 and fat metabolism in aquatic animal which has not been reported. .In this project, we will select the orange-spotted grouper, Epinephelussp coioides as research species. The grouper were developed by omprehensive breeding technique as a new variety with fast growth, high market value, and are considered a commercially important teleost for inland culture in China.Using RNA interference，real time fluorescence quantitative PCR technology, Western Blotting analysis combined with modern aquatic animal nutrition, we will focus on the following: ①studying the effects of dietary VA levels on mRNA and protein expression of RBP4 and fat metabolism key enzymes in liver for the grouper; ②examining the influences of VA levels on activities and mRNA expression of fat metabolism key enzymes were studied, before and after RBP4 gene silencing,respectively, in liver for the grouper..Through the studies on the relationship between VA and the RBP4 mRNA and protein expression in liver, we will clarify the relationship between the VA and the fat metabolism. In this projects, we will aim at the basics of the molecular metabolism of VA in grouper, as well as controlling mode of the VA for fat metabolism in the liver. These studies will have a important impact upon the sustainable development of aquaculture, and shed light on the resolution of the nutritional fatty liver problems which affect the seawater aquaculture.

脂肪肝问题制约海水鱼养殖的健康发展，而营养不平衡则是导致脂肪肝形成的重要因素。以期通过营养调控措施进行预防与控制脂肪肝形成，已成为水产动物营养研究的热点之一。哺乳动物研究发现，新型脂肪源信号因子—视黄醇结合蛋白4（RBP4）对脂肪代谢起重要调节作用，而RBP4合成与分泌与维生素A（VA）密切相关。目前，关于VA与RBP4及脂肪代谢的相互关系在鱼类未见报道。因此，本项目以斜带石斑鱼为对象，运用体外细胞培养实验、RNA干扰、实时荧光定量PCR和蛋白印迹技术并结合现代水产动物营养学方法，主要研究：①VA对肝脏RBP4 mRNA和蛋白表达的影响；②RBP4基因沉默前后，VA对脂肪代谢关键酶活性及其mRNA表达的影响；从而明确VA与RBP4及脂肪代谢之间的相互关系，为阐明VA介导RBP4参与肝脏脂肪代谢的调控机制奠定基础，同时也为解决石斑鱼等海水鱼类出现营养性脂肪肝现象提供新的思路。

脂肪肝问题是制约海水鱼养殖的健康发展，而营养不平衡则是导致脂肪肝形成的重要因素。以期通过营养调控措施进行预防与控制脂肪肝形成，已成为水产动物营养研究的热点之一。在哺乳动物研究中发现，新型脂肪源信号因子—视黄醇结合蛋白4（RBP4）对脂肪代谢起着重要调节作用，而RBP4合成与分泌与维生素A（VA）密切相关。目前，关于VA与RBP4及脂肪代谢的相互关系在鱼类未见报道。因此，本项目以斜带石斑鱼为对象，运用体外细胞培养实验、RNA干扰、实时荧光定量PCR和蛋白印迹技术并结合现代水产动物营养学方法，研究VA对肝脏RBP4 mRNA和蛋白表达的影响；明确VA与RBP4及脂肪代谢之间的相互关系，为阐明VA介导RBP4参与肝脏脂肪代谢的调控机制奠定基础，同时也为解决石斑鱼等海水鱼类出现营养性脂肪肝现象提供新的思路。. 研究结果发现：（1）维生素A缺乏抑制了鱼类的生长、抗氧化、脂质代谢过程和消化能力以及肠道形态的发育；（2）维生素A缺乏通过减少LZY、C3、C4、AKP、ACP和抗菌肽等先天性免疫因子，上调与IκBα/NF-κB（P65和c-Rel）信号分子相关的促炎细胞因子，下调鱼类抗炎细胞因子，从而抑制了肠道免疫功能；（3）维生素A缺乏可能通过下调紧密连接蛋白（claudin3和occludin）的mRNA水平来抑制紧密连接功能，从而影响鱼类物理屏障；（4）维生素A缺乏会抑制有益菌的丰度，增加有害菌的丰度。最后，根据WG和肠道LYZ活性的折线模型，珍珠龙胆石斑鱼对维生素A的需要量分别为2688.58和4096.36 IU/kg。