14-3-3γ蛋白协同mTOR信号通路影响奶牛乳腺上皮细胞生理功能

doi:10.13523/j.cb.20150606

中国生物工程杂志

2015, Vol. 35

Issue (6): 32-39 DOI: 10.13523/j.cb.20150606

研究报告

14-3-3γ蛋白协同mTOR信号通路影响奶牛乳腺上皮细胞生理功能

刘静¹, 骆超超², 黄建国², 吴迪¹, 高学军², 刘玉芬¹

1. 哈尔滨师范大学生命科学与技术学院哈尔滨 150080;
2. 东北农业大学农业生物功能基因重点实验室哈尔滨 150030

14-3-3γ Regulates the Physiological Function of Dairy Cow Mammary Gland Epithelial Cells Through mTOR Signaling Pathway

LIU Jing¹, LUO Chao-chao², HUANG Jian-guo², WU Di¹, GAO Xue-jun², LIU Yu-fen¹

1. College of Life Science and Technology, Harbin Normal University, Harbin 150080, China;
2. Key Laboratory of Dairy Science of Education Ministry, Northeast Agricultural University, Harbin 150030, China

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摘要：

14-3-3γ作为蛋白质与蛋白质之间的"桥梁蛋白",广泛参与细胞凋亡、细胞分裂、信号转导和蛋白质合成等生物体所有的重要生命活动的调节。以体外培养的荷斯坦奶牛(Vaccas)乳腺上皮细胞(dairy cow mammary gland epithelial cells, DCMECs)为模型,研究14-3-3γ对奶牛乳腺上皮细胞乳蛋白、乳脂合成的影响。瞬时和稳定转染pGCMV/IRES/EGFP/14-3-3γ即14-3-3γ过表达的奶牛乳腺上皮细胞,应用CASY细胞活力分析仪分析细胞活力的变化,测定细胞中甘油三脂(triglyceride, TG)和β-酪蛋白(β-Casein,CSN2)的合成,并且通过Western blot检测DCMECs中mTOR(mammalian target of rapamycin, mTOR)和p-mTOR的表达量,探究14-3-3γ影响奶牛乳腺上皮细胞生理功能的机制。结果显示,瞬时和稳定转染pGCMV/IRES/EGFP/ 14-3-3γ 的奶牛乳腺上皮细胞相对正常培养的奶牛乳腺上皮细胞,细胞活力极显著增强(P<0.01),CSN2和TG表达量极显著增加(P<0.01),同时mTOR和p-mTOR的表达量也显著增加(P<0.01)。研究结果表明14-3-3γ参与泌乳关键信号通路即mTOR信号通路,能够增强乳腺上皮细胞活力,促进奶牛乳腺上皮细胞CSN2和TG的合成,丰富了奶牛泌乳信号通路,为乳品质研究提供了新的理论依据。

关键词： 14-3-3γ 奶牛; 乳腺上皮细胞; mTOR; 信号通路

Abstract:

As a linker of proteins, 14-3-3γ plays critical roles in cell signal transductions, cell growth,differentiation and protein synthesis. Based on cultured dairy cow mammary gland epithelial cells in vitro, the impact of 14-3-3γ on the physiological function of dairy cow mammary gland epithelial cells were ivestigated. Expressing plasmids of 14-3-3γ gene were constructed, then stably and transiently transfected into dairy cow mammary gland epithelial cells. The effect of over-expression on dairy cow mammary gland epithelial cells was analyzed by CASY. Triglyceride was detected by triglyceride GPO-POD assay and beta casein was detected by high performance liquid chromatography. Expression of mTOR and p-mTOR was detected by Western blot. Experimental results show that the viability of dairy cow mammary gland epithelial cells, the expression of triglyceride and β-casein, the expression of mTOR and p-mTOR was identified up-expressed(P<0.01). All above results indicated that 14-3-3γ regulates the physiological function of dairy cow mammary gland epithelial cells through mTOR signaling pathway. The result enriches the lactation signaling pathway, which provides theoretical basis for improving the quality of milk.

Key words: 14-3-3γ Dairy cow Mammary gland epithelial cells mTOR Signaling pathway

收稿日期: 2015-02-11 出版日期: 2015-06-25

ZTFLH:

Q819

基金资助:

国家"973"计划(2011CB100804)、国家"863"计划(2013AA102504-03)资助项目

通讯作者: 刘玉芬 E-mail: liuyufen0825@126.com

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引用本文:

刘静, 骆超超, 黄建国, 吴迪, 高学军, 刘玉芬. 14-3-3γ蛋白协同mTOR信号通路影响奶牛乳腺上皮细胞生理功能[J]. 中国生物工程杂志, 2015, 35(6): 32-39.

LIU Jing, LUO Chao-chao, HUANG Jian-guo, WU Di, GAO Xue-jun, LIU Yu-fen. 14-3-3γ Regulates the Physiological Function of Dairy Cow Mammary Gland Epithelial Cells Through mTOR Signaling Pathway. China Biotechnology, 2015, 35(6): 32-39.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20150606 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I6/32

[1] 曾妍, 郑志红, 杨磊. 14-3-3蛋白功能研究进展. 现代肿瘤医学,2008,1(7): 1236-1238. Zeng Y, Zheng Z H, Yang L. Advance in 14-3-3 proteins function. Modern Oncology, 2008,1(7): 1236-1238.

[2] Lam T, Thomas L M, White C A, et al. Scaffold functions of 14-3-3 adaptors in B cell immunoglobulin class switch DNA recombination. Plos One, 2013,8(11): 80141.

[3] Zhao J, Meyerkord C L, Du Y, et al. 14-3-3 proteins as potential therapeutic targets. Semin Cell Dev Biol, 2011,22(7): 705-712.

[4] Zhao J, Du Y, Horton J R, et al. Discovery and structural characterization of a small molecular 14-3-3 protein-protein interaction inhibitor. Proc Natl Acad Sci USA, 2012,108(39): 16212-16216.

[5] 刘丹, 尹东, 廖章萍, 等. p38MAPK/14-3-3γ通路在LPS预处理对抗心肌细胞缺氧/复氧损伤中的作用. 中国药理学通报,2012,28(9):1239-1243. Liu D, Yin D, Liao Z P, et al. Role of p38 MAPK/14 -3 -3 γ signal pathway in LPS preconditioning against cardiomyocytes anoxia / reoxygenation injury. Chinese Pharmacological Bulletin, 2012,28(9):1239-1243.

[6] 程道宾, 陈小武. 14-3-3γ过表达通过抑制LRRK2 的去磷酸化防护鱼藤酮诱导的多巴胺能细胞损伤. 中风与神经疾病杂志, 2013,30(8): 703-706. Cheng D B, Chen X W. Overexpression of 14-3-3γ protects rotenone induced PC12 cells injury through preventing dephosphorylation of LRRK2.J Apoplexy and Nervous Diseases, 2013,30(8): 703-706.

[7] 沈奇,张文文, 陶雪娇, 等. 14-3-3γ表达载体的构建及其对子宫肌瘤细胞增殖和凋亡的影响. 温州医科大学学报, 2014,44(2): 79-82. Shen Q, Zhang W W, Tao X J, et al. Construction of a 14-3-3γ expression vector and its effect on proliferation and apoptosis of uterine leiomyoma cells. Journal of Wenzhou Medical University, 2014,44(2): 79-82.

[8] Dong Y, Zhao R, Chen X Q, et al. 14-3-3γ and neuroglobin are new intrinsic protective factors for cerebral ischemia. Mol Neurobiol, 2010,41(2-3):218-231.

[9] Burgos S A, Dai M, Cant J P. Nutrient availability and lactogenic hormones regulate mammary protein synthesis through the mammalian target of rapamycin signaling pathway. Journal of Dairy Science, 2010,93(1): 153-161.

[10] Rius A G, Appuhamy J A D R N, Cyriac J, et al. Regulation of protein synthesis in mammary glands of lactating dairy cows by starch and amino acids. Journal of Dairy Science, 2010,93(7): 3114-3127.

[11] Tong H L, Li Q Z, Gao X J, et al. Establishment and characterization of a lactating dairy goat mammary gland epithelial cell line. In Vitro Cell Dev Biol Anim, 2012,48(3): 149-155.

[12] Lu L M, Li Q Z, Huang J G, et al. Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis. Moleculaes, 2013,18(1): 263-275.

[13] Wang L N, Lin Y, Bian Y J, et al. Leucyl-tRNA synthetase regulates lactation and cell proliferation via mTOR signaling in dairy cow mammary epithelial cells. Int J Mol Sci, 2014,15(4): 5952-5969.

[14] Li H M, Wang C M, Li Q Z, et al. MiR-15a decreases bovine mammary epithelial cell viability and lactation and regulates growth hormone receptor expression. Molecules, 2012,17(10): 12037-12048.

[15] 陈洪菊, 屈艺,母得志. mTOR信号通路的生物学功能. 生命的化学, 2010,30(4):555-561. Chen H J, Li J H, Mu D Z. The progress of study on the biological function of mTOR pathway.Chemistry of Life, 2009,30(4):555-561.

[16] Sancak Y, Bar-Peled L, Zoncu R, et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids.Cell,2010,141(2):290.303.

[17] 文捐, 庹勤慧. SREBP通过调节脂质代谢影响细胞生长. 生命的化学. 2011,31(6): 790-795. Wen J, Tuo Q H. SREBP affect cell growth by regulating lipid metabolism. Chemistry of Life, 2011,31(6):790-795.

[18] Sege R R, Krebs E G. The MAPK signaling cascade. The FASEB Journal,1995, 9(9): 726-735.

[19] Ma X M, Blenis J. Molecular mechanisms of mTOR mediated translational control. Nature Reviews Molecular Cell Biology, 2009,10(5): 307-318.

[20] Zheng Q, Yin G, Yan C, et al. 14-3-3β binds to big mitogen-activated protein kinasel (BMK1 /ERK5) and regulates BMK1 function. The Journal of Biological Chemistry, 2004,279(10): 8787-8791.

[21] Li G, White C A, Lam T, et al. Combinatorial H3K9acS10ph histone modifications in IgH locus S regions target 14-3-3 adaptors and AID to specify antibody class-switch DNA recombination. Cell Reports, 2013,5(3): 702-714.

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