KISS-1/GPR54系统对奶牛乳腺上皮细胞合成乳蛋白的影响及其机制

Low nutritional quality of cow's milk has increasingly become a severe challenge faced by the healthy development of dairy industry in China. Milk protein is the main substance to determine the nutritional quality of cow's milk. Cow's milk quality could be affected by many factors, where neuroendocrine system plays an important regulatory role to protein contents in cow's milk. KISS-1/GPR54 system is a neuroendocrine regulation system with various biological functions. Considering its irreplaceable regulatory role in cancer metastasis and reproduction, research on endocrinology has always focused on the KISS-1/GPR54 system. In previous study, we found out that the KISS-1/GPR54 system may regulate the dairy cow’s mammary epithelial cell protein synthesis. The project will use western blotting, real-time PCR, ELISA, immunofluorescence, laser scanning confocal microscopy, liquid scintillation counting and other methods to explore the function of amino acid uptake, calcium ion concentr, cell polarity, and the synthesis and secretion of milk protein in mammary epithelial cells so as to identify the effects and molecular mechanism of KISS-1/GPR54 system on protein synthesis of cow's milk from molecular, cellular and overall levels. These results will provide scientific basis to further study the regulatory mechanism of protein synthesis of cow's milk and improve the nutritional quality of cow's milk.

乳蛋白是构成牛奶营养品质的主要物质基础，乳蛋白含量是奶业核心竞争力的重要标志。神经内分泌系统对奶牛乳蛋白合成具有重要的调控作用。KISS-1/GPR54系统是一种具有多种生物学功能的神经内分泌调控系统，是当前神经内分泌生理学研究的热点之一。前期研究发现，KISS-1/GPR54系统可能具有调控奶牛乳腺上皮细胞乳蛋白合成和分泌的重要作用。本项目拟利用Western blot、RT-PCR、ELISA、细胞免疫荧光、激光共聚焦显微镜、液体闪烁计数等技术，研究KISS-1/GPR54系统对乳腺上皮细胞氨基酸摄取功能、钙离子浓度、细胞极性变化、乳蛋白合成情况及相关信号分子的影响，并进行在体验证，从分子、细胞和整体水平上明确KISS-1/GPR54系统对奶牛乳腺上皮细胞合成乳蛋白的影响及机制，以期为深入认识奶牛乳蛋白合成的调控机理提供科学依据，为通过人工干预手段来提高牛奶营养品质奠定理论基础。

本项目对KISS-1/GPR54系统促进奶牛乳蛋白合成进行深入研究，并全面阐述了KISS-1/GPR54系统对乳质提升的机制。首先我们收集高乳品质奶牛乳腺和低乳品质奶牛乳腺组织，通过分析乳腺组织中GPR54和CSN2的表达情况，发现GPR54和CSN2的表达呈显著正相关。在奶牛乳腺上皮细胞中，Kp-10能显著促进CSN2基因和蛋白的表达，而GPR54抑制剂显著抑制Kp-10的这种作用。Kp-10通过GPR54激活奶牛乳腺上皮细胞中ERK1/2、AKT、mTOR和STAT5信号通路，而抑制ERK1/2、AKT、mTOR和STAT5磷酸化则抑制Kp-10诱导的CSN2的合成。乳脂和乳蛋白一样是评价乳品质的关键指标。在奶牛乳腺上皮细胞中，我们研究发现Kp-10通过GPR54抑制SIRT6的表达以维持mTOR信号通路的活化从而促进乳脂的合成。乳腺上皮细胞的数量和活性反应乳腺的泌乳能力，乳腺上皮细胞增殖和凋亡之间的动态平衡影响乳腺的泌乳能力。促进乳腺上皮细胞增殖有助于提高乳腺的泌乳量。我们研究发现Kp-10通过GPR54活化ERK1/2和AKT/mTOR信号以及促进RB-E2F1复合物解离，进而促进奶牛乳腺上皮细胞增殖。这一结果在体外培养的乳腺组织块中得到了进一步验证。此外，体内注射Kp-10也可显著促进青春期小鼠乳腺发育。以上结果提示Kp-10通过多种方式提高乳品质。这些结果为深入研究乳合成调控机制和乳腺发育提供了科学依据，为通过人工干预手段来提高牛奶营养品质奠定了理论基础。