动物营养学报 >

2016 , Vol. 28 >Issue 3: 916 - 925

DOI: https://doi.org/10.3969/j.issn.1006-267x.2016.03.034

研究简报 Short Communications

组氨酸对体外培养奶牛乳腺上皮细胞β-酪蛋白及酪氨酸激酶2-信号转导与转录激活子5/哺乳动物雷帕霉素靶蛋白信号通路相关磷酸化蛋白表达的影响

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  • 1. 内蒙古农业大学动物科学学院, 呼和浩特 010018;
    2. 农业部奶及奶制品质量监督检验测试中心(北京), 北京 100193;
    3. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193
王珊珊(1990-),女,黑龙江哈尔滨人,硕士研究生,动物营养与饲料科学专业。E-mail:imauwss@163.com

收稿日期: 2015-10-13

  网络出版日期: 2016-03-14

基金资助

奶业"973"计划(2011CB100800);现代农业产业技术体系专项资金(nycytx-04-01)

收起

Effects of Histidine on Expressions of β-casein, and Janus Kinases 2-Signal Transducer and Activator of Transcription 5/Mammalian Target Rapamycin Signaling Pathways Related Phospho-Proteins of in Vitro Cultured Bovine Mammary Epithelial Cells

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  • 1. College of Animal Science, Inner Mongolia Agricultural University, Hohhot 010018, China;
    2. Ministry of Agriculture-Milk and Dairy Product Inspection Center, Beijing 100193, China;
    3. State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2015-10-13

  Online published: 2016-03-14

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摘要

本试验旨在研究不同浓度的组氨酸对体外培养奶牛乳腺上皮细胞β-酪蛋白及酪氨酸激酶2(JAK2)-信号转导与转录激活子5(STAT5)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路相关磷酸化蛋白表达的影响。将原代奶牛乳腺上皮细胞进行体外培养,分为对照组和7个试验组,采用无必需氨基酸的培养基,对照组不添加组氨酸,试验组是在对照组基础上分别添加0.15、0.60、1.20、2.40、4.80、9.60、19.20 mmol/L的组氨酸。采用噻唑蓝比色法检测原代奶牛乳腺上皮细胞12 h增殖情况;运用蛋白质免疫印迹检测β-酪蛋白和8个信号通路相关磷酸化蛋白表达。结果表明:1)当组氨酸浓度为0.15~9.60 mmol/L时,与对照组相比,奶牛乳腺上皮细胞数量均增加。2)β-酪蛋白表达量随组氨酸浓度的增加出现先升高后降低的趋势,但试验组均极显著高于对照组(P<0.01)。3)与对照组相比,组氨酸的添加可极显著促进各信号通路相关磷酸化蛋白的表达(P<0.01);试验组中,随着组氨酸浓度的升高,磷酸化哺乳动物雷帕霉素靶蛋白[P-mTOR(Ser2481)]和磷酸化真核细胞翻译延伸因子2[P-eEF2(Thr56)]蛋白的表达量下降,而磷酸化核糖体S6蛋白激酶1[P-S6K1(Thr389)]的蛋白表达增加;当组氨酸浓度为2.40 mmol/L时,磷酸化酪氨酸激酶2[P-JAK2(Tyr1007/1008)]、磷酸化真核细胞始动因子4E结合蛋白1[P-4EBP1(Thr37)]和磷酸化真核细胞起始因子2α[P-eIF2α(Ser51)]蛋白的表达量最高,磷酸化信号转导与转录激活子5[P-STAT5(Tyr694)]、磷酸化mTOR调控蛋白[P-raptor(Ser863)]和mTOR复合物1中的绑定蛋白(GβL)蛋白在组氨酸浓度为9.60 mmol/L时表达量最高。综合可知,组氨酸的添加可通过促进JAK2-STAT5信号通路中P-JAK2(Tyr1007/1008)和P-STAT5(Tyr694)蛋白的表达来进而调控β-酪蛋白表达。最适浓度(0.15~9.60 mmol/L)范围内的组氨酸还可通过mTORC1的P-raptor(Ser863)蛋白作用于下游靶点P-4EBP1(Thr37)来促进β-酪蛋白表达,最终调控乳蛋白合成。

关键词： 组氨酸; 细胞增殖; 酪氨酸激酶2; 哺乳动物雷帕霉素靶蛋白; &beta; -酪蛋白

本文引用格式

王珊珊, 高海娜, 赵圣国, 郑楠, 张养东, 王加启, 闫素梅 . 组氨酸对体外培养奶牛乳腺上皮细胞β-酪蛋白及酪氨酸激酶2-信号转导与转录激活子5/哺乳动物雷帕霉素靶蛋白信号通路相关磷酸化蛋白表达的影响[J]. 动物营养学报, 2016 , 28(3) : 916 -925 . DOI: 10.3969/j.issn.1006-267x.2016.03.034

Abstract

The aim of this study was to investigate the effects of supplementation of different concentrations of histidine on expressions of β-casein and Janus kinases 2 (JAK2)-signal transducer and activator of transcription 5 (STAT5)/mammalian target rapamycin (mTOR) signaling pathways related phospho-proteins of in vitro cultured bovine mammary epithelial cells. The primary bovine mammary epithelial cells were used in this study, the special culture medium without essential amino acid (EAA) was used. Supplementation levels of histidine were 0 (control), 0.15, 0.60, 1.20, 2.40, 4.80, 9.60 and 19.20 mmol/L, respectively. After cultured for 12 h, primary bovine mammary epithelial cells proliferation was dectected by thiazolyl blue (MTT) method, the expressions of β-casein and 8 signaling pathway related phospho-proteins were determined by Western-Blot. The results showed as follows: 1) compared with control group the bovine mammary epithelial cell proliferation was increased after the supplementation of 0.15 to 9.60 mmol/L of histidine. 2) With the increase histidine concentration, the expression of β-casein measured tended to be increased at first and then decrease, and all experimental groups were significantly higher than control group (P<0.01). 3) Compared with control group, the supplementation of histidine could significantly promote the expressions of signaling pathway related phospho-proteins (P<0.01); among experimental groups, the expressions of phospho-mTOR [P-mTOR (Ser2481)] and phospho-eukaryotic translation elongation factor 2 [P-eEF2 (Thr56)] were reduced along with the increasing histidine concentration, and the expression of phospho-ribosome protein subunit 6 kinase 1 [P-S6K1 (Thr389)] was increased with the increasing histidine concentration; the expression of phospho-JAK2 [P-JAK2(Tyr1007/1008)], phospho-eukaryotic initiation factor 4E binding protein [P-4EBP1(Thr37)] and phospho-eukaryotic initiation factor 2α [P-eIF2α (Ser51)] were the highest at the supplementation of 2.40 mmol/L histidine, and the expressions of phospho-STAT5 [P-STAT5(Tyr694)], phospho-mTOR raptor [P-raptor(Ser863)] and mTOR complex binding protein (GβL) were highest at the supplementation of 9.60 mmol/L histidine. These results demonstrate that histidine can promote the expression of β-casein via P-JAK2(Tyr1007/1008) and P-STAT5(Tyr694) of JAK2-STAT5 signaling pathway. Histidine at optimal concentration (0.15 to 9.60 mmol/L) range can also promote the expression of β-casein via P-raptor (Ser863) of mTORC1, which act on down-stream targets P-4EBP1 (Thr37), and finally regulation of milk protein synthesis.

Key words： histidine; cell proliferation; JAK2; mTOR; β-casein

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