亮氨酸对乳蛋白合成影响及其机制的研究

A Study of Effect of Leucine on Kappa Casein Synthesis and Mechanism

该研究旨在探讨其他氨基酸缺乏条件下补充亮氨酸对体外培养的奶牛乳腺上皮细胞κ-酪蛋白基因表达和κ-酪蛋白合成的影响以及细胞增殖的影响,并从m RNA水平探究了m TOR信号通路介导的乳腺上皮细胞内蛋白质合成的重要性。为创造细胞氨基酸缺乏环境,将DMEM/F12培养基稀释100倍作为对照组,处理组1在对照组的基础上补充亮氨酸,处理组2在处理组1的基础上添加对m TOR信号通路上游PI3K起抑制作用的LY294002抑制剂,分别采用实时荧光定量PCR技术和酶联免疫法测定基因的相对表达量和蛋白合成量,采用CCK-8法测定细胞增殖。结果发现,在其他氨基酸缺乏的条件下,单独的亮氨酸显著促进了κ-酪蛋白基因表达和κ-酪蛋白合成,而添加抑制剂极显著降低了亮氨酸的这种作用;蛋白质合成信号通路相关的基因m TOR、S6K1、e IF4E、e EF2相对表达量均以处理组1最高,处理组2最低,但除了e EF2基因外,其他基因在各组间差异不显著;4EBP1的基因以处理组1最高,对照组最低;补充亮氨酸没有显著促进乳腺上皮细胞增殖,但添加抑制剂可显著抑制细胞增殖。结果表明,在其他氨基酸缺乏的条件下给奶牛乳腺上皮细胞单一补充亮氨酸能显著促进κ-酪蛋白的合成,这种作用可能与m TOR信号通路介导蛋白质合成有关,此外,研究也表明m TOR信号通路可调控奶牛乳腺上皮细胞的增殖。

This study was conducted to investigate the effects of leucine on proliferation of bovine mammary epithelial cells and κ-casein gene expression and protein synthesis in bovine mammary epithelial cells cultured in vitro under nutrition-deficient conditions and the importance of protein synthesis in mammary epithelial cells mediated by m TOR signaling pathway at m RNA level. To creat a lack of amino acids environment, DMEM/F12 medium diluted 100-fold was used as the control group. Treatment group 1 was based on the control group supplemented with leucine and treatment group 2 was added with LY294004 to inhibite PI3 K on the basis of the treatment group 1. Real-time PCR, ELISA and CCK-8 methods were used to determine gene relative expression, protein synthesis and cell proliferation, respectively. The results show that expression level of CSN3 gene and κ-casein were increased by leucine significantly, however, inhibitor LY294002 weakened its roles dramatically. Expression of the genes（m TOR, S6K1, e IF4 E, e EF2 and 4EBP1） related to protein synthesis of treatment group 1 were the highest while of the treatment group 2 were the lowest, however, the differences were not significant except for e EF2. Gene expression was the highest in treatment group 1 and the lowest in control group for 4EBP1. Proliferation of the cells supplemented with leucine was not significantly changed, but it was decreased by inhibitor LY294002 observably. In conclusion, leucine has a stimulatory effect on CSN3 gene and κ-casein expression while any other amino acids are deprived and this role may be relate to the synthesis of protein mediated by m TOR pathway. In addition, the results also indicated that m TOR pathway can regulate the proliferation of bovine mammary epithelial cells.