应用三维细胞培养模型研究脂肪酸和激素对奶牛乳腺上皮细胞乳脂肪合成的影响及分子机理

The low milk quality is one of limiting factor of dairy industry development in China. Increasing milk fat content and improving fatty acids composition can promote the overall development level of dairy industry. Milk fat precursors and neuroendocrine play a major role in milk fat synthesis, however the effect of fatty acids and hormones on milk fat synthesis and regulation mechanisms are still not clear. Previous studies about milk components synthesis in bovine mammary gland epithelial cell are almost conducted by two-dimensional cultivation model, however due to loss of acinar-like structures, results obtained from two-dimensional cultivation model can not really reflect mammary gland metabolism in vivo. In this research subject, three-dimensional cultivation model will be applied to study the effect of short chain, medium chain fatty acids, unsaturated fatty acids with eighteen carbon atoms and hormones on milk fat synthesis in bovine mammary gland epithelial cells, so as to reveal the ideal substrates mode which can promote milk fat synthesis and improve milk fatty acids composition. Meanwhile, the molecular mechanisms of milk fatty acids and hormones regulation milk fat synthesis will be clarified through genes expression of transcriptional regulation factors, genes associated with fatty acids and lipid synthesis, and Akt signal molecule. Protein expression of transcriptional regulation factors involved in milk fat synthesis will also be tested. Results of this subject can provide theory basis for directional regulation of milk fat synthesis.

乳品质低下是目前我国奶业发展的瓶颈，提高乳脂肪含量、改善乳脂肪酸组成，对于提升我国奶业整体发展水平，促进奶业健康发展具有重要意义。脂肪酸和激素是调控乳脂合成的重要因素，然而脂肪酸和激素对乳脂合成的影响及其作用机制仍不明确。国内外研究工作者目前关于乳成分合成的体外研究大多是以二维培养的细胞模型开展的，而二维模型由于缺失了乳腺上皮细胞的极化结构，使得研究结果不能真实反映活体内乳腺组织的代谢情况。为此，本项目以三维培养的奶牛乳腺上皮细胞为模型，研究中短链脂肪酸、18碳不饱和脂肪酸和激素对乳脂肪合成的影响，揭示促进乳脂肪合成、改善乳脂肪酸组成的理想底物供应模式；并从乳脂肪合成和转运相关基因表达，转录调控因子基因表达、蛋白表达，Akt信号通路等方面，阐明脂肪酸和激素对奶牛乳腺内乳脂肪合成的调控机理，为定向调控乳脂肪合成提供理论依据。

本研究建立了成熟的奶牛乳腺上皮细胞三维培养模型。在三维培养的奶牛乳腺上皮细胞培养液中单一或组合添加短链脂肪酸（乙酸钠和丁酸钠）、长链脂肪酸（油酸、亚油酸和亚麻酸）和泌乳相关激素（催乳素、胰岛素和生长激素），研究其对奶牛乳腺上皮细胞乳脂肪和乳蛋白合成的影响，并从脂肪合成转录调控因子PPARγ和SREBP1代谢网络、蛋白合成mTOR信号通路、JAK/STAT5信号通路转录组学角度阐释了脂肪酸和激素影响奶牛乳腺上皮细胞乳脂肪和乳蛋白合成的机理，探明了促进奶牛乳腺上皮细胞乳脂肪和乳蛋白合成的理想脂肪酸和泌乳激素供给模式。研究结果表明：（1）短链脂肪酸组合添加时，乙酸钠:β-羟丁酸钠=2:1，即乙酸钠和β-羟丁酸钠添加浓度分别为9.6 mmol/L 和4.8 mmol/L 对奶牛乳腺上皮细胞乳脂肪和乳蛋白合成促进效果较好。（2）长链脂肪酸组合添加时，油酸:亚油酸:亚麻酸=2:13.3:1, 即油酸、亚油酸和亚麻酸的添加量分别为17.3 μmol/L、115.05 μmol/L和8.65 μmol/L对奶牛乳腺上皮细胞乳脂肪和乳蛋白合成促进效果较好。（3）泌乳相关激素组合添加时，催乳素:胰岛素:生长激素=10:1:3, 即催乳素、胰岛素和生长激素剂量分别为100 ng/mL、10 ng/mL和30 ng/mL对奶牛乳腺上皮细胞乳脂肪和乳蛋白的合成有较好的促进作用。（4）短链、长链脂肪酸和泌乳激素组合添加时，乙酸钠:β-羟丁酸钠=2:1, 油酸:亚油酸:亚麻酸=2:13.3:1，催乳素:胰岛素:生长激素=10:1:3,即乙酸钠、β-羟丁酸钠、油酸、亚油酸、亚麻酸、催乳素、胰岛素、生长激素的添加浓度分别为4.8 mmol/L、2.4 mmol/L、8.65 µmol/L、57.52 µmol/L、4.32 µmol/L、50 nmol/L、5 nmol/L和15 nmol/L时对 BMECs 乳脂肪和乳蛋白的合成有较好的促进作用。（5）三维培养模式下单一或组合添加脂肪酸和激素 BMECs 内TAG的合成量、乳脂肪和乳蛋白合成相关基因的表达量均显著高于二维培养模式。上述研究成果为调控乳成分合成，改善乳品质提供了理论依据。