脂肪酸对奶牛乳腺上皮细胞乳蛋白和乳脂合成相关基因表达量的影响

Effects of Fatty Acids on Expression Levels of Genes Involved in Milk Protein and Milk Fat Synthesis in Bovine Mammary Epithelial Cells

本试验旨在探寻促进奶牛乳腺上皮细胞(BMECs)乳蛋白和乳脂合成的短链脂肪酸(乙酸、β-羟丁酸)和长链脂肪酸(油酸、亚油酸、亚麻酸)的组合添加模式,为调控乳成分合成提供理论依据。BMECs经分离、纯化后,选取第2代细胞,分为5组,对照组不添加脂肪酸,Ⅰ组和Ⅱ组添加的乙酸、β-羟丁酸浓度比例均为2.0(9.60 mmol/L)∶1.0(4.80 mmol/L),油酸、亚油酸、亚麻酸的浓度比例分别为2.0(17.30μmol/L)∶13.3(115.05μmol/L)∶1.0(8.65μmol/L)和9.6(75.20μmol/L)∶7.4(58.00μmol/L)∶1.0(7.80μmol/L);Ⅲ组和Ⅳ组添加的乙酸、β-羟丁酸的浓度比例均为1.0(7.20 mmol/L)∶1.0(7.20 mmol/L),油酸、亚油酸、亚麻酸的浓度比例分别为2.0∶13.3∶1.0和9.6∶7.4∶1.0,各组添加的短链脂肪酸(SCFA)和长链脂肪酸(LCFA)总浓度为14.541 mmol/L,每组3个重复。培养24 h后,检测细胞相对增殖率(RGR)、甘油三酯(TAG)的合成量以及乳蛋白和乳脂合成相关基因的表达量。结果表明:1)试验组BMECs RGR及TAG的合成量均显著高于对照组(P<0.05);Ⅰ组RGR最高,TAG合成量最多。2)与对照组相比,Ⅱ组显著提高了核糖体蛋白S6激酶1(S6K1)、κ-酪蛋白(CSN3)基因的表达量(P<0.05);Ⅳ组显著提高了CSN3、蛋白激酶B(AKT)、S6K1、真核翻译起始因子4E结合蛋白1(4EBP1)基因的表达量(P<0.05);试验组信号转导和转录激活因子5(STAT5)基因的表达量显著降低(P<0.05)。3)与对照组相比,试验组二酰甘油酰基转移酶2(DG AT2)基因的表达量显著提高(P<0.05),脂肪酸合成酶(FASN)基因的表达量显著降低(P<0.05)。综上所述,在培养液中添加7.20 mmol/L乙酸、7.20 mmol/Lβ-羟丁酸、75.20μmol/L油酸、58.00μmol/L亚油酸、7.80μmol/L亚麻酸对BM ECs乳蛋白和乳脂合成相关基因的表达量有较好的促进作用。

The aim of this study w as to explore combined supplemental model of short chain fatty acids（ acetic acid,β-hydroxybutyric acid） and long chain fatty acids（ oleic acid,linoleic acid,linolenic acid） to promote milk protein and milk fat synthesis in bovine mammary epithelial cells（ BMECs）,to provide theoretical basis for the regulation of synthesis of milk composition. BMECs w ere separated and purified,and the second generation of cells w as selected and divided into 5 groups w ith 3 replicates per group. No fatty acid w as added in control group; in groups Ⅰ and Ⅱ,the concentration radio of supplied acetate acid and β-hydroxybutyric acid w as 2.0（ 9.60 mmol/L） ∶1.0（ 4.80 mmol/L）,how ever,the concentration radio of oleic acid,linoleic acid and linolenic acid w as 2. 0（ 17. 30 μmol/L） ∶ 13. 3（ 115. 05 μmol/L） ∶ 1. 0（ 8. 65 μmol/L） and 9. 6（ 75.20 μmol/L） ∶7.4（ 58.00 μmol/L） ∶1.0（ 7.80 μmol/L）,respectively; in groups Ⅲ and Ⅳ,the concentration radio of acetate acid and β-hydroxybutyric acid w as 1. 0 ∶ 1. 0,how ever,the concentration radio of oleic acid,linoleic acid and linolenic acid w as 2.0∶13.3∶1.0 and 9.6∶7.4∶1.0,respectively. The total concentration of short chain fatty acids and long chain fatty acids w as 14.541 mmol/L. After cultured for 24 h,relative grow th rate（ RGR）,triglyceride（ TAG） synthesis amount and expression levels of genes involved in milk protein and milk fat synthesis w ere measured. The results show ed as follow s： 1） RGR and TAG synthesis amount of BMECs in experimental groups w ere significantly higher as comparing w ith control group（ P 0.05）;RGR and TAG synthesis amount w ere the highest in group Ⅰ. 2） Compared w ith control group,expression levels of ribosomal protein S6 kinase（ S6 K1） and κ-casein（ CSN3） genes in group Ⅱ w ere significantly increased（ P 0.05）; expression levels of CSN3,protein kkinase B（ AKT）,S6 K1 and eukaryotic initiation 4 E binding protein（ 4 EBP1） gene in group Ⅳ w as significantly increased（ P 0. 05）; expression level of signal transduction and transcriptional activation factor 5（ STAT5） gene in experimental groups w as significantly decreased（ P 0. 05）. 3） Compared w ith control group,expression level of diacylgycerol acyltransferase 2（ DGAT2） gene in experimental groups was significantly increased（ P〈0.05）,and expression level of fatty acid synthase（ FASN） gene w as significantly decreased（ P 0.05）. In conclusion,expression levels of genes involved in milk protein and milk fat synthesis has a better effect w hen culture medium is added w ith7.20 mmol/L acetate acid,7. 20 mmol/L β-hydroxybutyric acid,75. 20 μmol/L oleic acid,58. 00 μmol/L linoleic acid,and 7.80 μmol/L linolenic acid.