T10,c12-CLA通过PI3K/akt信号通路调控奶山羊乳腺脂肪酸合成的分子机制

The project is going through the methods (Co-immunoprecipitation(Co-IP),Western blot,Immunofluorescence,Real time RT-PCR, gas chromatography-mass spectrometry and so on) with Xinong Saanen Goats as experimental object. After dairy goat mammary gland epithelial cells are treated by t10,c12-CLA, cell cycle and the mark genes of proliferation and apoptosis are detected, and the expressions and interrelationships and phosphorylation levels of p85 and PDK1/2 and Akt in PI3K/akt signal pathway are analyzed. Through the preparation of Akt gene over-expression and RNA interference vectors which are transfected to dairy goat mammary gland epithelial cells,the effects of t10,c12-CLA on the expressions of vital genes of fatty acid synthesis are investigated. Then,the effects of t10,c12-CLA on fatty acid synthesis and mammary gland structures are studied in adult diary goat through employing abomasal t10,c12-CLA infusion and t10,c12-CLA supplement in the ration.The researches will certify the mechnisms of t10,c12-CLA regulating fatty acid synthesis of dairy goat mammary gland by PI3K/akt signal pathway in theory, and the results of which provide theoretical evidences for improving milk flavor and quality and have a positive impaction on developing goat milk industry through t10,c12-CLA supplement in goat's feed.

本课题以西农莎能奶山羊为研究对象，采用Co-IP、Western blot、免疫荧光、实时定量PCR及气相色谱-质谱联用等技术，检测经反10,顺12共轭亚油酸（ t10,c12-CLA）处理后羊乳腺上皮细胞增殖凋亡标记基因及细胞周期变化，PI3K/akt信号通路关键因子p85、PDK1/2、Akt的表达量变化、相互作用及磷酸化水平。构建奶山羊Akt基因真核表达载体和特异性RNAi载体并转染羊乳腺上皮细胞，研究t10,c12-CLA对脂肪酸合成关键基因表达的影响。采用真胃灌注及日粮添加t10,c12-CLA，在成体水平上观察t10,c12-CLA调节乳腺脂肪酸合成及对乳腺组织的影响。该研究将从理论上明确t10,c12-CLA通过PI3K/akt信号通路对羊乳腺脂肪酸合成的调控机制，为改善羊乳风味、提高羊乳品质提供理论参考，并将通过饲料添加t10,c12-CLA对羊奶产业发展产生积极影响。

动物乳中脂肪酸种类丰富，尤其是短、中链脂肪酸对人体有益，但也是形成羊乳特殊风味（膻味）的主要因素。通过改变这类脂肪酸在羊乳中含量，就能从羊体本身限制或消除这种特殊风味产生的源头，改善羊乳口感。共轭亚油酸（CLA）是一种天然活性脂肪酸，具有调节羊乳脂肪酸含量的作用。本课题以西农莎能奶山羊为研究对象，采用Westernblot、免疫荧光、实时定量PCR、气相色谱-质谱联用、高通量测序等试验技术，通过培养奶山羊乳腺上皮细胞及建系，分析t10,c12-CLA对奶山羊乳腺上皮细胞增殖与凋亡的影响、参与PI3K/akt信号通路的调控，以及对奶山羊乳腺上皮细胞中脂肪酸代谢的影响，同时通过日粮添加等手段分析t10,c12-CLA对乳脂合成的影响机制，解析t10,c12-CLA通过PI3K/akt信号通路调控奶山羊乳腺脂肪酸合成的分子机制。本项目研究首先分离获得了奶山羊乳腺上皮细胞，将pCI-neo-hTERT载体转染于奶山羊乳腺上皮细胞得到了能行使正常功能的奶山羊乳腺上皮细胞系；通过EGF、PRLR基因以及Akt1基因相关试验研究证实PI3K/akt信号通路可明显上调脂肪酸代谢相关基因并导致细胞内甘油三脂的变化；体内试验证实t10,c12-CLA影响羊乳脂肪酸组成并明显抑制脂肪酸合成、转运、活化以及甘油三脂合成、分泌基因等的表达，这些生理过程均归因于t10,c12-CLA 对PI3K/akt信号通路的抑制，与此同时发现t10c12-CLA通过影响AMPK通路也参与脂肪酸的调控，明晰了t10,c12-CLA调控奶山羊乳腺上皮细胞脂肪酸合成的分子事件，为通过 t10,c12-CLA调控羊乳中脂肪酸代谢提供新的理论支持，并对奶山羊饲料添加剂研究提供新的思路。