SIRT1/FOXO1通路在奶山羊乳腺脂肪酸代谢中的调控机理

Investigating the regulation of fatty acid metabolism in goat mammary gland is of the vital scientific significance on improving goat milk flavor and quality. The mammary epithelial cells are isolated and cultured from dairy goat. The cells are treated with agonists and inhibitors of SIRT1. RT-qPCR, western blot and gas chromatography-mass spectrometry technology are used to determine the composition of fatty acid and expression of fatty acid metabolism genes. Through the preparation of SIRT1 gene over-expression and RNA interference vectors which are transfected to dairy goat mammary epithelial cells, the composition of fatty acid and expression of fatty acid metabolism genes are detected. Then, the promoter of ATGL and FASN are cloned and predicted by bioinformatics. The SIRT1/FOXO1 acting element of promoter are researched using deletion mutants, site-directed mutagenesis, double luciferase reporter gene activity detection and CHIP technology. The researches will certify the mechnisms of regulating fatty acid metabolism of dairy goat mammary gland by SIRT1/FOXO1 signal pathway in theory, and the results of which provide theoretical and experimental basis for manipulating goat milk fatty acids content, and improving the goaty flavor.

研究乳腺脂肪酸代谢调控对改善羊奶风味、提高羊奶品质具有重要科学意义。本课题以奶山羊为研究对象，分离培养奶山羊乳腺上皮细胞，用SIRT1激动剂和抑制剂处理细胞，SIRT1/FOXO1通路激活或抑制后，采用实时定量PCR、Western blot及气相色谱-质谱联用技术，检测细胞中脂肪酸组成和脂肪酸代谢基因表达情况。构建SIRT1基因过表达和干扰载体，同时干扰FOXO1基因，检测乳腺上皮细胞脂肪酸组成及脂肪酸代谢关键基因的表达。克隆奶山羊FASN和ATGL基因启动子，通过生物信息学预测启动子上的调控元件，同时采用缺失突变、定点突变、双报告基因活性检测及CHIP技术研究启动子上SIRT1/FOXO1通路的作用元件。该研究将从理论上明确SIRT1/FOXO1信号通路对奶山羊乳腺脂肪酸代谢的调控机制，为调控羊奶脂肪酸含量和改善羊奶风味等提供理论和实验依据。

奶山羊乳腺脂肪酸代谢受诸多因素的调控。SIRT1基因是具有乙酰化作用的沉默信息调控子，SIRT1表达可升高FOXO1的去乙酰化水平，在哺乳动物体内起到抑制脂肪生成的作用。本研宄以奶山羊乳腺上皮细胞为研宄对象，探讨SIRT1/FOXO1对奶山羊乳腺细胞脂质代谢及脂肪酸组成的影响。结果发现，SIRT1/FOXO1通路激活显著下调乳脂关键调控因子SREBP1和PPARγ表达，脂肪酸合酶FASN、脂肪酸去饱和酶SCD1表达量显著下降，而脂解基因ATGL表达显著上调；激活SIRT1/FOXO1通路抑制了细胞中甘油三酯合成，脂滴聚积减少，同时改变了细胞中脂肪酸组成，C16:0和C18:1的比例显著降低，C18:0的比例显著增多。反之，SIRT1/FOXO1通路抑制后，对细胞中脂质积累及脂肪酸组成具有相反作用。随后，通过过表达SIRT1基因，结果发现，过表达SIRT1抑制 SREBP1蛋白表达；同时抑制细胞中脂滴的聚积和甘油三酯合成，细胞中C16:0脂肪酸比例显著降低。反之，干扰SIRT1促进了细胞中脂质积累。进一步研究发现，SIRT1/FOXO1通路抑制FASN启动子活性，并且是通过抑制SREBP1实现的，在野生型FASN启动子中过表达SREBP1之后可以显著减弱SIRT1/FOXO1导致的FASN启动子活性降低。SIRT1/FOXO1上调ATGL启动子活性，PPARγ干扰后减弱了SIRT1/FOXO1对ATGL启动子的上调作用，表明SIRT1/FOXO1通过PPARγ调控ATGL启动子活性。这些结果明确了SIRT1/FOXO1信号通路对奶山羊乳腺细胞脂肪酸代谢的调控机制，为调控羊奶脂肪酸组成和改善羊奶风味、提高羊奶品质具有重要意义。