亮氨酸水平对奶牛乳腺上皮细胞增殖及&kappa;-酪蛋白合成相关基因表达的影响

代文婷, 李爱军, 郑楠, 王加启

doi:10.3969/j.issn.1006-267x.2015.05.028

动物营养学报 >

2015 , Vol. 27 >Issue 5: 1559 - 1566

DOI: https://doi.org/10.3969/j.issn.1006-267x.2015.05.028

研究简报 Short Communications

亮氨酸水平对奶牛乳腺上皮细胞增殖及κ-酪蛋白合成相关基因表达的影响

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1. 吉林大学动物科技学院, 长春 130062;
2. 中国农业科学院北京畜牧兽医研究所, 农业部奶产品质量安全风险评估实验室(北京), 北京 100193;
3. 农业部奶及奶制品质量监督检验测试中心 (北京), 北京 100193;
4. 唐山市畜牧水产品质量监测中心, 唐山 063000

代文婷(1988—),女,山西临汾人,硕士研究生,从事奶牛乳蛋白合成调控研究。E-mail:daiwenting_2012@sina.com

收稿日期: 2014-12-10

网络出版日期: 2015-05-13

基金资助

国家重点基础研究发展计划(2011CB100805);现代农业产业技术体系专项资金(nycytx-04-01);中国农业科学院科技创新工程(ASTIP-IAS12)

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Effects of Leucine Level on Proliferation and κ-Casein Synthesis Related Gene Expressions of Bovine Mammary Epithelial Cells

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1. College of Animal Science and Technology, Jilin University, Changchun 130062, China;
2. Ministry of Agriculture Laboratory of Quality & Safety Risk Assessment for Dairy Products (Beijing), Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
3. Ministry of Agriculture-Milk and Dairy Product Inspection Center (Beijing), Beijing 100193, China;
4. The Livestock and Aquatic Products Quality Inspection and ITest Center (Tangshan), Tangshan 063000, China

Received date: 2014-12-10

Online published: 2015-05-13

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摘要

本试验旨在研究添加不同水平亮氨酸对乳腺上皮细胞体外增殖及κ-酪蛋白(CSN3)合成相关基因表达的影响。选用中国荷斯坦奶牛乳腺上皮细胞进行体外培养,以不含有亮氨酸的培养基为对照组(0×组),试验组培养基分别添加0.112 5(0.25×组)、0.225 0(0.5×组)、0.450 0(1×组)、0.900 0(2×组)、1.800 0(4×组)、3.600 0(8×组)、7.200 0(16×组)、14.400 0 mmol/L(32×组)的亮氨酸。试验采用噻唑蓝(MTT)比色法检测奶牛乳腺上皮细胞增殖;运用实时定量PCR检测CSN3合成相关基因相对表达量。结果表明:亮氨酸能够提高乳腺上皮细胞的相对增殖率。24和48 h时,除0.25×组外,其余各组与对照组相比细胞均差异均显著(P<0.05);72 h时,各组与对照组相比差异均显著(P<0.05);其中2×组的促增殖作用都达到最强。亮氨酸能够促进哺乳动物雷帕霉素靶蛋白(mTOR)、核糖体蛋白S6激酶1(S6K1)、蛋白质酪氨酸激酶2(JAK2)、转录激活因子5(STAT5)和CSN3基因的表达,抑制真核细胞翻译启动因子4E结合蛋白1(4EBP1)基因的表达,其中2×组对mTOR、S6K1、JAK2、STAT5和CSN3基因的上调表达最强,而对4EBP1基因的下调表达最强。总之,添加亮氨酸能够促进奶牛乳腺上皮细胞增殖和CSN3合成相关基因(除4EBP1)表达,亮氨酸水平为0.900 0 mmol/L时,其促进作用均达到最大。

关键词： 亮氨酸; 细胞增殖; κ-酪蛋白; 基因表达

本文引用格式

代文婷, 李爱军, 郑楠, 王加启 . 亮氨酸水平对奶牛乳腺上皮细胞增殖及κ-酪蛋白合成相关基因表达的影响[J]. 动物营养学报, 2015 , 27(5) : 1559 -1566 . DOI: 10.3969/j.issn.1006-267x.2015.05.028

Abstract

This experiment was conducted to investigate the effects of the supplementation of leucine (Leu) in culture medium at different levels on proliferation and κ-casein (CSN3) synthesis related gene expressions of bovine mammary epithelial cells (BMECs). BMECs of Chinese Holstein dairy cows were used in this study. Supplementation levels of Leu were 0 (0×group, control group), 0.112 5 (0.25×group), 0.225 0 (0.5×group), 0.450 0 (1×group), 0.900 0 (2×group), 1.800 0 (4×group), 3.600 0 (8×group), 7.200 0 (16×group), 14.400 0 mmol/L (32×group), respectively. The proliferation of BMECs was determined using thiazolyl blue (MTT) method, and the relative expression levels of CSN3 synthesis related gene were determined using real-time PCR. The results showed as follows: the relative proliferation rate of BMECs was promoted by different levels of Leu. Compared with control group, the promotion efficiency of all the experimental groups was significantly higher except 0.25× group after incubation for 24 and 48 h (P<0.05), and the promotion efficiency of all the experimental groups was significantly higher after incubation for 72 h (P<0.05). After incubation for 24, 48 and 72 h, the promotion efficiency of 2×group was the highest. The relative expression levels of mTOR, S6K1, JAK2, STAT5 and CSN3 genes were increased by Leu, and that of 4EBP1 gene was decreased. The upper-/down-regulated expression levels reached the highest/lowest in 2×group. In summary, Leu can promote cell proliferation and CSN3 synthesis related gene expressions of BMECs, and the promotion efficiency is the highest when Leu level is 0.900 0 mmol/L.

Key words： leucine; cell proliferation; κ-casein; gene expression

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