维生素A和乙酸交互作用对奶牛乳腺上皮细胞中乳成分合成相关基因表达的影响  

Effect of Interaction Between Vitamin A and Acetic Acid on the Expression of Genes Related to Milk Composition Synthesis in Bovine Mammary Epithelial Cells

作　　者：郭咏梅[1] 刘阳 武汭 闫素梅[1] 赵艳丽[1] 郭晓宇[1] GUO YongMei;LIU Yang;WU Rui;YAN SuMei;ZHAO YanLi;GUO XiaoYu(College of Animal Science,Inner Mongolia Agricultural University/Key Laboratory of Animal Nutrition and Feed Science at Universities of Inner Mongolia Autonomous Region,Hohhot 010018)

机构地区：[1]内蒙古农业大学动物科学学院/内蒙古自治区高校动物营养与饲料科学重点实验室,呼和浩特010018

出　　处：《中国农业科学》2023年第21期4344-4358,共15页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31160466);内蒙古农业大学高层次人才科研启动项目(NDYB2020-4);内蒙古农业大学基本科研业务费专项资金(BR220142);内蒙古自治区本级事业单位引进优秀人才科研基金。

摘　　要：【目的】在前期研究发现维生素A(VA)对奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMECs)内乳脂和乳蛋白合成相关基因表达具有显著促进效果的基础上,以奶牛乳腺上皮细胞为研究对象,添加乳脂肪合成前体物乙酸(acetic acid,AA),旨在探究VA和AA二者在乳成分合成相关基因表达上是否存在互作关系,借此更系统地了解VA参与乳脂、乳蛋白合成的调控机制,为奶牛饲料中合理添加VA及改善乳品质提供科学依据。【方法】采用胶原酶消化法培养细胞,将第三代BMECs分为6个处理组,分别加入不同浓度的VA和AA混合工作液,6个处理组依次命名为对照组(0 mg·mL^(-1)VA+0 mol·L^(-1)AA)、AA处理1组(0 mg·mL^(-1)VA+0.006 mol·L^(-1)AA)、AA处理2组(0 mg·mL^(-1)VA+0.01 mol·L^(-1)AA)、VA处理组(0.001 mg·mL^(-1)VA+0 mol·L^(-1)AA)、VAAA1组(0.001 mg·mL^(-1)VA+0.006 mol·L^(-1)AA)和VAAA2组(0.001 mg·mL^(-1)VA+0.01 mol·L^(-1)AA)。培养结束后收集细胞和培养液样品,采用MTT法测定细胞增殖率;采用试剂盒法测定甘油三酯含量、乳脂和乳蛋白合成关键酶的活性;采用实时定量PCR技术测定并计算乳脂和乳蛋白合成相关基因的相对表达量。【结果】添加VA后显著提高了细胞增殖率(P<0.001),但添加AA、VA与AA的组合效应均对细胞增殖率无显著影响(P>0.05)。VA和AA在乳脂合成方面存在互作效应,AA能提高BMECs内的甘油三酯(TG)含量(P=0.01),同步添加VA则对TG的合成产生明显的抑制作用(P=0.01);AA能提高细胞内脂肪酸合成酶(FASN)、乙酰辅酶A羧化酶(ACACA)的活性及过氧化物酶体增殖物激活受体γ(PPARG)基因的表达(P<0.05;P<0.05;P<0.001),同时添加VA则极显著地抑制(P<0.001;P=0.01;P<0.01);AA显著提高了BMECs内哺乳动物雷帕霉素靶蛋白(mTOR)含量(P=0.05)、p70核糖体蛋白S6激酶1(S6K1)酶活性(P<0.001),并上调了κ-酪蛋白(CSN3)的基因表达(P<0.001),mTOR与β-酪蛋白(CSN2)的基因表达受到两者互�【Objective】Our previous research found that vitamin A(VA)has a significant effect on promoting the expression of genes related to milk fat and milk protein synthesis in bovine mammary epithelial cells(BMECs).Based on this,this experiment was conducted by adding acetic acid(AA)to explore whether there is an interaction between VA and AA in the expression of genes related to milk composition synthesis,then gaining a more systematic understanding of the regulatory mechanism of VA involvement in milk fat and protein synthesis,this study would provide scientific basis for the rational addition of VA to feed for dairy cows and the improvement of milk quality.【Method】In this experiment,cells were cultured using digestive method of collagenase.Two factor completely randomized trial design was used in this study.The third generation BMECs was randomly divided into 6 treatment groups,with 6 replicates in each group.After starvation treatment of serum-free medium for 24 hours,medium with VA and AA at different concentrations was added,respectively.The six treatment groups were sequentially named as control group(0 mg·mL^(-1)VA+0 mol·L^(-1)AA),AA treatment group 1(0 mg·mL^(-1)VA+0.006 mol·L^(-1)AA),AA treatment group 2(0 mg·mL^(-1)VA+0.01 mol·L^(-1)AA),VA treatment group(0.001 mg·mL^(-1)VA+0 mol·L^(-1)AA),VAAA1 group(0.001 mg·mL^(-1)VA+0.006 mol·L^(-1)AA),and VAAA2 group(0.001 mg·mL^(-1)VA+0.01 mol·L^(-1)AA).After 24 hours of continuous culture,the cells and culture medium were collected as required,and the cell proliferation rate was measured using MTT method;the content of triglycerides and the activities of key enzymes in milk fat and protein synthesis were measured using kits.The relative expression of genes related to milk fat and milk protein synthesis was measured and calculated by real-time quantitative PCR.【Result】The results showed that adding VA significantly increased the cell proliferation rate(P<0.001),but no significant effect on the cell proliferation rate was found followed by additio

关 键 词：维生素A 乙酸 互作 奶牛乳腺上皮细胞 乳成分 

分 类 号：S823[农业科学—畜牧学]