亮氨酸對乳蛋白合成影響及其機制的研究

龐學燕　王洪榮

摘 要：該研究旨在探討其他氨基酸缺乏條件下補充亮氨酸對體外培養的奶牛乳腺上皮細胞κ-酪蛋白基因表達和κ-酪蛋白合成的影響以及細胞增殖的影響，并從mRNA水平探究了mTOR信號通路介導的乳腺上皮細胞內蛋白質合成的重要性。為創造細胞氨基酸缺乏環境，將DMEM/F12培養基稀釋100倍作為對照組，處理組1在對照組的基礎上補充亮氨酸，處理組2在處理組1的基礎上添加對mTOR信號通路上游PI3K起抑制作用的LY294002抑制劑，分別采用實時熒光定量PCR技術和酶聯免疫法測定基因的相對表達量和蛋白合成量，采用CCK-8法測定細胞增殖。結果發現，在其他氨基酸缺乏的條件下，單獨的亮氨酸顯著促進了κ-酪蛋白基因表達和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；蛋白質合成信號通路相關的基因mTOR、S6K1、eIF4E、eEF2相對表達量均以處理組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，對照組最低；補充亮氨酸沒有顯著促進乳腺上皮細胞增殖，但添加抑制劑可顯著抑制細胞增殖。結果表明，在其他氨基酸缺乏的條件下給奶牛乳腺上皮細胞單一補充亮氨酸能顯著促進κ-酪蛋白的合成，這種作用可能與mTOR信號通路介導蛋白質合成有關，此外，研究也表明mTOR信號通路可調控奶牛乳腺上皮細胞的增殖。

關鍵詞：乳腺上皮細胞 CSN3 κ-酪蛋白 mTOR 基因表達 細胞增殖

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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