精氨酸调控珍珠龙胆石斑鱼肌卫星细胞激活的分子机制

Our previous study showed that the dietary arginine significantly affected pearl gentian grouper growth. Muscle satellite cell activation is the first step in muscle fiber proliferation and determines fish growth. In this project, different dietary levels of arginine feeding trial and BrdU injection experiments will be first conducted in pearl gentian grouper, and through analysis muscle satellite cell cycle changes and MRFs genes expression to determine dietary arginine on satellite cell activation. Furthermore, based on vitro muscle satellite cell culture experiment, arginine and TOR inhibitor will be added in the medium, through immunochemical analysis of Pax7\MyoD cell localization and expression and satellite cell activation to study if TOR involved in arginine activation of muscle satellite cells. Finally, the Co-IP and mass spectrometry techniques will be used to isolate and identify the regulatory role of TOR-dependent transcription factors on MyoD gene transcription and verified by luciferase reporter and RNAi essay. In summary, this project will clarify cellular and molecular mechanisms of arginine in the regulation of muscle satellite cell activation and initially uncover the potential mechanism of arginine regulation in fish growth.

我们前期研究表明饲料精氨酸显著影响珍珠龙胆石斑鱼生长。肌卫星细胞激活是肌纤维增殖的首要环节，在鱼体生长中起到决定作用。由此本项目将首先通过不同精氨酸水平饲料养殖实验，利用BrdU注射和流式细胞仪检测肌卫星细胞激活情况，结合生肌调节因子（MRFs）基因表达变化，确定精氨酸对珍珠龙胆石斑鱼肌卫星细胞的激活作用。其次通过体外肌卫星细胞培养，向培养基中添加精氨酸和TOR抑制剂后检测肌卫星细胞激活情况，并利用免疫化学分析Pax7和MyoD细胞定位及表达水平，研究TOR是否参与精氨酸对肌卫星细胞激活调节作用并且影响MyoD基因表达。最后借助免疫共沉淀和质谱技术手段分离鉴定TOR依赖性蛋白因子对MyoD转录表达调节作用，并通过荧光素酶报告基因和RNAi验证候选蛋白因子与MyoD相互作用。以期从细胞和分子水平阐明精氨酸调控珍珠龙胆石斑鱼肌卫星细胞激活作用机制，初步揭示精氨酸调控鱼体生长潜在原因。

鞍带石斑鱼常作为石斑鱼杂交的父本，以获取其快速生长的优良性状，因此其是研究鱼类肌肉生长发育的良好模型。骨骼肌的生长发育对于鱼类生长至关重要，而肌卫星细胞增殖和分化所形成的肌纤维是其主要组成部分。因此本项目建立了鞍带石斑鱼的肌卫星细胞培养体系，以期为后续深入研究鱼类骨骼肌发育的营养调控分子机制奠定良好基础。同时，利用RACE技术，在鞍带石斑鱼中克隆得到了生肌调节因子家族MyoD（MyoD1和MyoD2）、MyoG、MRF4、Myf5全长cDNA序列；且通过基因表达实验发现精氨酸有可能通过调控生肌调节因子的表达从而促进鱼体生长。本项目相关研究成果将有利于进一步深入探究并阐明营养素（氨基酸）如何调控鱼类生长发育的潜在分子机制。