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  

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

作　　者：代文婷[1,2,3] 李爱军 郑楠[2,3] 王加启[1,2,3] 

机构地区：[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

出　　处：《动物营养学报》2015年第5期1559-1566,共8页CHINESE JOURNAL OF ANIMAL NUTRITION

基　　金：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)

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(m TOR)、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m TOR、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。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 thia- zolyl 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 ef- ficiency of all the experimental groups was significantly higher after incubation for 72 h （P〈0.05）. After incu- bation for 24, 48 and 72 h, the promotion efficiency of 2 ×group was the highest. The relative expression levels of roTOR, S6K1, JAK2, STATS and CSN3 genes were increased by Leu, and that of 4EBP1 gene was de- creased. 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.

关 键 词：亮氨酸 细胞增殖 κ-酪蛋白 基因表达 

分 类 号：S823[农业科学—畜牧学]