奶牛乳腺对含限制性氨基酸小肽的摄取利用机制研究

Dairy milk is a main source of protein in human diet. Milk protein content is a very important index for milk quality evaluation and price assessment. Thus improvement of dairy milk protein content and yield has always been an important area for researchers and dairy industries. Free amino acids are the major form in which amino-nitrogen is taken up by lactating mammary tissues. However, this concept has been challenged for a long time. In lactating dairy cows, the uptake of certain essential amino acids within the mammary gland appears to be insufficient for milk protein synthesis. The amino acids that are in short supply for milk protein synthesis requirement appear to activate their withdrawal from the peptide-bound amino acids pool. The utilization of circulating peptides for protein synthesis appears to be a general phenomenon. The mechanisms for uptake and utilization of small peptides in mammary gland are complicated and still unclear. Therefore, the objective of this project is to evaluate the molecular and cellular mechanisms of uptake and utilization of limiting amino acids-containing small peptides in bovine mammary gland using mammary epithelial cell culture model, arteriovenous difference, isotope kinetic and biopsy techniques. Knowledge of the absorption and utilization of small peptides in mammary gland may provide new insights into protein metabolism and secretion by the mammary gland and provide scientific basis for the application of small peptides in dairy cows.

乳蛋白作为人类食品蛋白的重要来源及牛乳质量、收购定价的重要指标，其重要性一直受到生产者和研究者的广泛关注，提高奶牛乳产品特别是乳蛋白产量及质量意义重大。鉴于乳腺组织能够利用小肽合成乳蛋白的观点已逐渐被人们接受和认可，结合目前反刍动物小肽吸收利用机制研究现状，本课题拟利用奶牛乳腺上皮细胞体外培养模型，结合同位素示踪、乳腺动静脉血插管和活体取样技术，在细胞、分子和组织多水平研究奶牛乳腺对含限制性氨基酸小肽的吸收利用机制及其对乳蛋白合成、分泌的调控规律，揭示小肽供应、吸收与乳蛋白合成调控间的关系，阐明奶牛乳腺对小肽的摄取利用机制。该研究可为构建奶牛泌乳需要的适宜小肽营养模型、完善乳腺蛋白质、氨基酸营养模式，定向调控乳成分提供理论依据，并为奶牛生产中小肽饲料的添加和新型蛋白质饲料的开发提供参考。

研究表明小肽在促进乳蛋白合成中具有重要作用但潜在机制尚未清晰。本项目旨在研究Met-Met在奶牛乳腺中的吸收特性以及其促进乳蛋白合成的分子机制。结果发现奶牛乳腺上皮细胞对β-Ala-Lys-AMCA和Met-Met-FITC的摄取呈现时间效应且呈现出浓度依赖性且符合米氏方程。Met-Gly、Lys-Lys、Gly-Met、Gly-Leu和Met-Leu显著性抑制Met-Met和β-Ala-Lys-AMCA的吸收。His对β-Ala-Lys-AMCA的吸收没有影响。siRNA干扰PepT2后，β-Ala-Lys-AMCA和Met-Met的吸收显著下降。Met-Met呈现剂量依赖性的促进β-CN和PepT2的表达，Met-Met 最适添加量为80 µg/ml且最适替代比例为15%。抑制PepT2可显著降低αs1-CN的表达。与照组相比15％Met-Met组AA转运载体ATB0,+ 及AA吸收率显著增加。Met-Met的添加显著提高了细胞活力，增强了细胞周期蛋白D1的表达。80 µg/ml Met-Met促进了S6K1和4E-BP1的磷酸化，且抑制mTOR和JAK2后Met-Met促进细胞活力和β-CN合成的效果显著下降。Met-Met替代Met时，仔鼠出生重、乳腺β酪蛋白表达和泌乳量显著增加。结果提示，PepT2在奶牛乳腺上皮细胞Met-Met摄取中发挥重要作用且Met-Met通过促进氨基酸供应和细胞增殖并激活JAK2-STAT5和mTOR促进乳蛋白的合成；Met-Met二肽较游离Met更有效的促进母鼠胚胎发育和泌乳性能，从而在后代生长发育中发挥重要作用。研究可为构建奶牛泌乳需要的适宜小肽营养模型、完善乳腺蛋白质、氨基酸营养模式，定向调控乳成分提供理论依据，并为奶牛生产中小肽饲料的添加和新型蛋白质饲料的开发提供参考。

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