亮氨酸對(duì)乳蛋白合成影響及其機(jī)制的研究

龐學(xué)燕　王洪榮

摘 要：該研究旨在探討其他氨基酸缺乏條件下補(bǔ)充亮氨酸對(duì)體外培養(yǎng)的奶牛乳腺上皮細(xì)胞κ-酪蛋白基因表達(dá)和κ-酪蛋白合成的影響以及細(xì)胞增殖的影響，并從mRNA水平探究了mTOR信號(hào)通路介導(dǎo)的乳腺上皮細(xì)胞內(nèi)蛋白質(zhì)合成的重要性。為創(chuàng)造細(xì)胞氨基酸缺乏環(huán)境，將DMEM/F12培養(yǎng)基稀釋100倍作為對(duì)照組，處理組1在對(duì)照組的基礎(chǔ)上補(bǔ)充亮氨酸，處理組2在處理組1的基礎(chǔ)上添加對(duì)mTOR信號(hào)通路上游PI3K起抑制作用的LY294002抑制劑，分別采用實(shí)時(shí)熒光定量PCR技術(shù)和酶聯(lián)免疫法測定基因的相對(duì)表達(dá)量和蛋白合成量，采用CCK-8法測定細(xì)胞增殖。結(jié)果發(fā)現(xiàn)，在其他氨基酸缺乏的條件下，單獨(dú)的亮氨酸顯著促進(jìn)了κ-酪蛋白基因表達(dá)和κ-酪蛋白合成，而添加抑制劑極顯著降低了亮氨酸的這種作用；蛋白質(zhì)合成信號(hào)通路相關(guān)的基因mTOR、S6K1、eIF4E、eEF2相對(duì)表達(dá)量均以處理組1最高，處理組2最低，但除了eEF2基因外，其他基因在各組間差異不顯著；4EBP1的基因以處理組1最高，對(duì)照組最低；補(bǔ)充亮氨酸沒有顯著促進(jìn)乳腺上皮細(xì)胞增殖，但添加抑制劑可顯著抑制細(xì)胞增殖。結(jié)果表明，在其他氨基酸缺乏的條件下給奶牛乳腺上皮細(xì)胞單一補(bǔ)充亮氨酸能顯著促進(jìn)κ-酪蛋白的合成，這種作用可能與mTOR信號(hào)通路介導(dǎo)蛋白質(zhì)合成有關(guān)，此外，研究也表明mTOR信號(hào)通路可調(diào)控奶牛乳腺上皮細(xì)胞的增殖。

關(guān)鍵詞：乳腺上皮細(xì)胞 CSN3 κ-酪蛋白 mTOR 基因表達(dá) 細(xì)胞增殖

Abstract：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 mTOR signaling pathway at mRNA 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 PI3K 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 （mTOR， S6K1， eIF4E， eEF2 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 eEF2. 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 mTOR pathway. In addition， the results also indicated that mTOR pathway can regulate the proliferation of bovine mammary epithelial cells.

Key Words：Mammary epithelial cells；CSN3；κ-casein；mTOR；Gene expression；Cell proliferation

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