精氨酸调控黄颡鱼肠道粘膜上皮细胞增殖的作用机制

Dietary L-arginine (Arg) can ameliorate intestinal morphology through accelerating intestinal mucosa development of mammalian. However, the effects of dietary L-arginine on intestinal development in fish have not been determined. And the metabolism and mechanism of L-arginine administration in fish intestinal metabolism and mechanism are not clear. Intestinal development is closely related to intestinal epithelial cell proliferation and differentiation. This project will be conducted to use intestinal epithelial cells model of Pelteobagrus fulvidraco in vitro primary cell culture that has been established in our prerious research work and to use molecular biology, cell biology, Western blotting and isotope labeling techniques. The metabolism path and transport rule of L-arginine administration in intestinal mucosal epithelial of Pelteobagrus fulvidraco will be investigated. Furthermore, the effects of L-arginine and its metabolites (NO and polyamines) on cell proliferation and differentiation, amino acid transporter (CAT1, CAT2, y+LAT1, y+LAT2) secretion, growth factor (EGF, EGF-R, IGF-1, IGF-1R) expression and cellular signaling pathways (mTOR and PI3k/Akt/Bcl2) will be elucidated. The results will help to reveal nutrition regulation mechanism of L-arginine on intestinal development in fish and to provide the scientific basis for the application of L-arginine in maintenance of fish intestinal health.

饲料添加精氨酸能够促进哺乳动物肠道粘膜发育和改善肠道形态结构。但精氨酸对水生动物黄颡鱼肠道发育在细胞和分子层面研究还未见报道，其在鱼类肠道中的代谢和作用机理尚不清楚。肠道发育与肠道粘膜上皮细胞增殖分化密切相关。本项目利用前期建立的黄颡鱼肠道粘膜上皮细胞原代培养模型，采用分子生物学、细胞生物学、蛋白质印迹和同位素标记等技术手段，研究精氨酸在黄颡鱼肠道粘膜上皮细胞的代谢路径和转运规律，探讨精氨酸及代谢产物（一氧化氮和多胺）对细胞增殖分化、氨基酸转运载体、细胞生长因子分泌表达及细胞信号通路的影响，阐明精氨酸调控肠道粘膜上皮细胞增殖的作用机制。研究结果为揭示精氨酸调控鱼类肠道生长发育的作用机制具有重要理论意义，同时为精氨酸在维护鱼类肠道健康的应用提供科学依据。

肠炎病是对鱼类危害最为严重的细菌性疾病之一，肠道健康对鱼类的生长至关重要，而饲料成分是影响肠道发育的重要因素。本项目采用动物营养学、动物生理学、分子生物学、细胞生物学等研究手段，细胞和分子水平研究了精氨酸对粘膜上皮细胞增殖的影响，构建了黄颡鱼肠道粘膜上皮细胞原代培养体系，培养液中精氨酸盐酸盐添加为1.6 g/L显著促进黄颡鱼肠道粘膜上皮细胞增殖，降低促炎因子分泌表达，下调蛋白质代谢关键基因如热休克蛋白（HSC70）mRNA、延长因子1a（EF1a）、核糖体L4（RPL4）mRNA相对表达量，揭示了精氨酸调控黄颡鱼肠道粘膜上皮细胞增殖、蛋白质代谢关键基因表达的作用机理，丰富了水产动物肠道健康营养调控理论。在此基础上，开展了黄颡鱼养殖试验进行验证，从整体和组织水平研究了精氨酸对黄颡鱼生长性能、肠道功能和肠道形态结构的影响，确定黄颡鱼饲料中精氨酸适宜需求量为2.74%（6.45%饲料蛋白），饲料精氨酸水平为2.81%显著升高黄颡鱼肠道蛋白酶和淀粉酶活性，增加肠道皱襞高度和肌层厚度，改善黄颡鱼肠道免疫功能，提高抗氨氮应激能力；添加480 mg/kg精氨酸显著提高黄颡鱼肠道皱壁高度、肌层厚度和杯状细胞数量，缓解氧化鱼油对黄颡鱼幼鱼生长的抑制作用，干预氧化鱼油对黄颡鱼幼鱼肠道免疫、抗氧化指标和组织结构的抑制作用。研究结果为揭示营养物质调控鱼类肠道生长发育的作用机制提供理论基础，同时为精氨酸在维护鱼类肠道健康的应用提供科学依据。