亮氨酸对奶牛乳腺上皮细胞内乳脂合成相关基因和蛋白表达的影响

摘要

本试验旨在研究亮氨酸(Leu)对泌乳奶牛乳腺上皮细胞(BMECs)内乳脂合成相关基因和蛋白表达的影响,以探讨Leu对乳脂合成的影响机理.将第3代BMECs随机分为6个处理,每个处理6个重复.6个处理培养液中Leu浓度分别为0.45、0.90、1.80、2.70、3.60和7.20 mmol/L,37℃、5%CO2培养48 h后测定BMECs内甘油三酯(TG)的含量及乳脂合成相关基因和过氧化物酶体增殖物激活受体γ(PPARγ)与固醇调节元件结合蛋白(SREBP1)蛋白的相对表达量.结果显示:Leu浓度对BMECs内TG含量无显著影响(P>0.05).适宜浓度的Leu显著促进脂肪酸合成酶(FASN)和乙酰辅酶A羧化酶A(ACACA)基因的表达(P<0.05),FASN基因的相对表达量以1.80~2.70 mmol/L Leu处理、ACACA基因的相对表达量以1.80~7.20 mmol/L Leu处理较高.Leu浓度显著影响BMECs内SREBP1基因及蛋白表达(P<0.05),以1.80 mmol/L Leu的促进效果最好.虽然Leu显著抑制BMECs内脂肪酸结合蛋白3(FABP3)、脂蛋白脂酶(LPL)、乙酰甘油磷酸脂酰转移酶6(AGPAT6)、线粒体甘油-3-磷酸酰基转移酶(GPAM)和嗜乳脂蛋白亚家族1成员1(BTN1A1)基因的表达(P<0.05),但只有高浓度(3.60~7.20 mmol/L)的Leu抑制作用较大.综合来看,Leu浓度影响BMECs乳脂合成相关基因及PPARγ和SREBP1蛋白的表达.Leu浓度为1.80~2.70 mmol/L时,对脂肪酸从头合成相关基因及调控因子SREBP1蛋白表达的促进效果较好,对TG合成及脂滴形成相关基因表达的抑制作用较小.%The objects of this study were to study the effects of leucine ( Leu) on expression of genes and pro-teins related with milk fat synthesis in bovine mammary epithelial cells ( BMECs) , in order to investigate the mechenism of Leu regulating milk fat synthesis. The third generation of BMECs were divided into six treat-ments with six replicates per treatment, and cultured in culture mediums with 0.45, 0.90, 1.80, 2.70, 3.60 and 7.20 mmol/L Leu, respectively. The triglyceride ( TG) content, the relative expression levels of genes re-lated with milk fat synthesis, as well as the protein relative expression levels of peroxisome proliferator-activa-ted receptor-γ( PPARγ) and sterol regulatory element binding protein 1 ( SREBP1) in BMECs after 48 h incu-bation at 37 ℃ and 5% CO2 were detected. The results showed that the Leu concentration had no significant effect on TG content in BMECs ( P>0.05) . The optimal Leu concentration significantly up-regulated the gene expression of fatty acid synthase (FASN) and acetyl-CoA carboxylase A (ACACA) in BMECs (P<0.05). The higher gene relative expression level of FASN was observed in 1.80 to 2.70 mmol/L Leu treatments, and the higher gene relative expression level of ACACA was observed in 1.80 to 7.20 mmol/L Leu treatments. Leu concentration significantly increased the gene and protein expression of SREBP1 in BMECs ( P<0.05) , and the best promoting effect was observed in 1. 80 mmol/L Leu treatment. Although Leu significantly inhibited the gene expression of fatty acid-binding protein 3 (FABP3), lipoprotein lipase (LPL), 1-acylglycerol-3-phos-phate O-acyltransferase 6 ( AGPAT6) , mitochondria glycerol-3-phosphate acyltrandferase ( GPAM) and buty-rophilin subfamily 1 member A1 (BTN1A1)in BMECs (P<0.05), the higher inhibiting effect was only ob-served in high Leu concentration (3.60 to 7.20 mmol/L) treatments. Taken together, Leu concentration has a significant effect on the expression of genes related with milk fat synthesis and protein expression of PPARγand SREBP1 in BMECs. The 1.80 to 2.70 mmol/L Leu has a better promoting effect on de novo fatty acid synthe-sis genes and SREBP1 protein expression, as well as little inhibiting effect on the expression of genes involved in TG synthesis and lipid droplet formation.