奶牛乳腺中4F2hc基因的表达模式及亮氨酸对其表达的调控研究

doi:10.16431/j.cnki.1671-7236.2020.08.005

摘要/Abstract

摘要： 为了研究4F2hc在奶牛乳腺中的表达模式及调控方式，进一步明确氨基酸在奶牛乳腺上皮细胞中的跨膜转运过程，本研究采用Western blotting和实时荧光定量PCR技术检测了4F2hc在泌乳期和干奶期奶牛乳腺组织中的表达变化；在体外培养的泌乳期奶牛乳腺上皮细胞中添加亮氨酸，采用Western blotting和实时荧光定量PCR技术检测其对奶牛乳腺上皮细胞中4F2hc表达的影响；采用雷帕霉素抑制剂抑制mTOR信号通路，使用Western blotting方法检测mTOR信号抑制后奶牛乳腺上皮细胞中4F2hc表达以及乳蛋白合成的变化。结果显示，在泌乳期的奶牛乳腺组织中4F2hc的mRNA和蛋白表达水平均显著或极显著高于干奶期（P<0.05，P<0.01）；在体外培养的奶牛乳腺上皮细胞中添加亮氨酸可以极显著提高乳腺上皮细胞中4F2hc的mRNA和蛋白质表达水平（P<0.01）；亮氨酸刺激可以激活细胞内的mTOR信号通路（P<0.05），而雷帕霉素处理则可以显著抑制mTOR信号分子的磷酸化并极显著抑制亮氨酸诱导的4F2hc的表达（P<0.05，P<0.01），进而极显著抑制β-Casein的合成（P<0.01）。以上研究结果表明，4F2hc基因的表达与奶牛乳腺的泌乳活性之间呈正相关，亮氨酸可以通过激活mTOR信号通路来调节4F2hc基因的表达，进而影响乳蛋白的合成。

关键词: 奶牛; 乳腺; 4F2hc基因; 泌乳

Abstract: To investigate the expression and regulation of 4F2hc in mammary glands of dairy cows,and clarify the process of amino acids transport across plasma membrane of mammary epithelial cells,the expression of 4F2hc inmammary tissues of lactation cows and dry cows were detected by Western blotting and Real-time quantitative PCR.The mammary epithelial cells of lactating cows in vitro were treated with leucine,the effect of leucine on the expression of 4F2hc in the mammary epithelial cells of lactating cows was also detected by Western blotting and Real-time quantitative PCR.The expression of 4F2hc and the synthesis of milk protein in the mammary epithelial cells of dairy cows were detected by Western blotting.The results showed that the mRNA and protein expression levels of 4F2hc were significantly or extremely significantly higher in mammary tissues of lactating cows than that in dry cows (P<0.05,P<0.01).Adding leucine to cultured bovine mammary epithelial cells could extremely significantly increase the expression of 4F2hc mRNA and protein (P<0.01).Leucine stimulation could activate the mTOR signaling pathway (P<0.05),while rapamycin treatment could significantly inhibit the phosphorylation of mTOR signaling molecules and the expression of 4F2hc induced by leucine (P<0.05,P<0.01),and then extremely significantly inhibit the synthesis of β-Casein(P<0.01).These results revealed that the expression of 4F2hc was positively correlated with lactation activity in mammary glands of dairy cows.Leucine could regulate 4F2hc expression via mTOR signaling pathway in mammary epithelial cells of dairy cows,thus affecting milk protein synthesis.

Key words: dairy cows; mammary gland; 4F2hc gene; lactation

中图分类号:

Q786

姜明慧, 黄鹤, 段晓宇, 江怡, 刘灿, 侯晓明. 奶牛乳腺中4F2hc基因的表达模式及亮氨酸对其表达的调控研究[J]. 中国畜牧兽医, 2020, 47(8): 2378-2384.

JIANG Minghui, HUANG He, DUAN Xiaoyu, JIANG Yi, LIU Can, HOU Xiaoming. Expression Pattern of 4F2hc Gene in Mammary Glands of Dairy Cows and Regulation of Its Expression by Leucine[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2378-2384.

参考文献

[1] MANJARIN R,BEQUETTE B J,WU G,et al.Linking our understanding of mammary gland metabolism to amino acid nutrition[J].Amino Acids,2014,46(11):2447-2462.
[2] 邢伟楠,段晓宇,杨洋,等.L型氨基酸转运载体1对奶牛乳腺中β-酪蛋白表达的影响[J].中国畜牧兽医,2016,43(10):2553-2559. XING W N,DUAN X Y,YANG Y,et al.Effect of L type amino acid transporter 1 on β-Casein expression in mammary epithelial cells of dairy cows[J].China Animal Husbandry & Veterinary Medicine,2016,43(10):2553-2559.(in Chinese)
[3] DUAN X,LIN Y,LV H,et al.Methionine induces LAT1 expression in dairy cow mammary gland by activating the mTORC1 signaling pathway[J].DNA and Cell Biology,2017,36(12):1126-1133.
[4] 林叶,侯晓明.L型氨基酸转运载体1及其配体在奶牛乳腺中的协同表达研究[J].畜牧兽医学报,2014,45(2):212-217. LIN Y,HOU X M.Research on L-type amino acid transporter 1 and its ligand coordinated expression in dairy cow mammary gland[J].Acta Veterinaria et Zootechnica Sinica,2014,45(2):212-217.(in Chinese)
[5] MATSUMOTO T,NAKAMURA E,NAKAMURA H,et al.Production of free glutamate in milk requires the leucine transporter LAT1[J].American Journal of Physiology-cell Physiology,2013,305(6):623-631.
[6] FINUCANE K A,MCFADDEN T B,BOND J P,et al.Onset of lactation in the bovine mammary gland:Gene expression profiling indicates a strong inhibition of gene expression in cell proliferation[J].Functional & Integrative Genomics,2008,8(3):251-264.
[7] YANG Y,LIN Y,DUAN X,et al.The effects of cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC) on milk lipid synthesis in mammary glands of dairy cows[J].Journal of Dairy Science,2017,100(5):4014-4024.
[8] HAYNES B F.Human T lymphocyte antigens as defined by monoclonal antibodies[J].Immunological Reviews,1981,57:127-161.
[9] FERAL C C,NISHIVA N,FENCZIK C A,et al.CD98hc (SLC3A2) mediates integrin signaling[J].Proceedings of the National Academy of Sciences of the United States of America,2005,102(2):355-360.
[10] PRAGER G W,FERAL C C,KIM C,et al.CD98hc (SLC3A2) interactionwith the integrin beta subunit cytoplasmic domain mediates adhesive signaling[J].Journal of Biological Chemistry,2007,282(33):24477-24484.
[11] FOTIADIS D,KANAI Y,PALACIN M.The SLC3 and SLC7 families of amino acid transporters[J].Molecular Aspects of Medicine,2013,34(2-3):139-158.
[12] LIN Y,DUAN X,LV H,et al.The effects of L-type amino acid transporter 1 on milk protein synthesis in mammary glands of dairy cows[J].Journal of Dairy Science,2018,101(2):1687-1696.
[13] LUO C,ZHAO S,DAI W,et al.Proteomic analyses reveal GNG12 regulates cell growth and casein synthesis by activating the Leu-mediated mTORC1 signaling pathway[J].Biochimica et Biophysica Acta-proteins and Proteomics,2018,1866(11):1092-1101.
[14] ZHAO Y,YAN S,CHEN L,et al.Effect of interaction between leucine and acetate on the milk protein synthesis in bovine mammary epithelial cells[J].Animal Science Journal,2019,90(1):81-89.
[15] REZAEI R,WU Z,HOU Y,et al.Amino acids and mammary gland development:Nutritional implications for milk production and neonatal growth[J].Journal of Animal Science and Biotechnology,2016,7:20.
[16] SHENNAN D B,CALVERT D T,TRAVERS M T,et al.A study of L-leucine,L-phenylalanine and L-alanine transport in the perfused rat mammary gland:Possible involvement of LAT1 and LAT2[J].Biochimica et Biophysica Acta,2002,1564(1):133-139.
[17] OHNO H,NAKATSU Y,SAKODA H,et al.4F2hc stabilizes GLUT1 protein and increases glucose transport activity[J].American Journal of Physiology-cell Physiology,2011,300(5):1047-1054.
[18] LAPLANTE M,SABATINI D M.Regulation of mTORC1 and its impact on gene expression at a glance[J].Journal of Cell Science,2013,126(Pt 8):1713-1719.
[19] KIMBALL S R,JEFFERSON L S.New functions for amino acids:Effects on gene transcription and translation[J].American Journal of Clinical Nutrition,2006,83(2):500S-507S.
[20] WANG C X,CHEN F,ZHANG W F,et al.Leucine promotes the growth of fetal pigs by increasing protein synthesis through the mTOR signaling pathway in longissimus dorsi muscle at late gestation[J].Journal of Agricultural and Food Chemistry,2018,66(15):3840-3849.
[21] LUO C,QI H,HUANG X,et al.GlyRS is a new mediator of amino acid-induced milk synthesis in bovine mammary epithelial cells[J].Journal of Cellular Physiology,2019,234(3):2973-2983.
[22] AO J,WEI C,SI Y,et al.Tudor-SN regulates milk synthesis and proliferation of bovine mammary epithelial cells[J].International Journal of Molecular Sciences,2015,16(12):29936-29947.
[23] APPUHAMY J A,KNOEBEL N A,NAYANANJALIE W A,et al.Isoleucine and leucine independently regulate mTOR signaling and protein synthesis in MAC-T cells and bovine mammary tissue slices[J].The Journal of Nutrition,2012,142(3):484-491.
[24] SUN J,LIU J,HUANG B,et al.Kisspeptin-10 induces β-Casein synthesis via GPR54 and its downstream signaling pathways in bovine mammary epithelial cells[J].International Journal of Molecular Sciences,2017,18(12):E2621.