赖氨酸蛋氨酸配比模式对奶牛乳腺上皮细胞酪蛋白合成的影响

Effect of Ratio between Lysine and Methionine on Casein Synthesis in Bovine Mammary Epithelial Cells

以体外培养的奶牛乳腺上皮细胞为模型,研究赖氨酸蛋氨酸配比模式对体外培养的奶牛乳腺上皮细胞酪蛋白合成的影响。试验采用因子试验设计,以蛋氨酸(0.4、0.5、0.6 mmol/L)和赖氨酸(1.0、1.2、1.4、1.5、1.6 mmol/L)的混合物作为试验处理。试验期为48 h,每个处理3个重复,试验重复3次。通过ELISA方法检测添加蛋氨酸和赖氨酸混合物后细胞中酪蛋白的合成量。得出最佳的赖氨酸蛋氨酸配比模式,并采用Real-Time PCR技术检测此水平下乳蛋白基因、与乳蛋白合成转录和翻译相关的基因以及与赖氨酸、蛋氨酸相关的转运载体CAT-1和ASCT-2的表达。结果表明:蛋氨酸(0.4 mmol/L)和赖氨酸(1.2 mmol/L)混合添加时奶牛乳腺上皮细胞总酪蛋白合成量最高为2.95 ppm,并差异极显著。当赖氨酸和蛋氨酸以1.2 mmol/L和0.4 mmol/L即3∶1混合添加时在不影响赖氨酸、蛋氨酸相关的转运载体CAT-1和ASCT-2基因表达的条件下均能极显著地促进乳蛋白基因的表达如:CSN1S1、CSN1S2、CSN2、CSN3和LALBA基因表达均上调,且CSN1S1和LABA表达量最高。与乳蛋白转录相关的STAT5-JAK2信号通路基因的表达均显著上调;与乳蛋白翻译相关的mTOR信号通路的相关基因除EIF4E-BP1显著下调,m TOR和S6K基因的表达均上调且mTOR差异极显著。

The experiment was conducted with a 5X3 factorial design： with lysine （1.0, 1.2, 1.4, 1.5, 1.6 mmolfL in DMEM//F12 medium） and methionine （0.4, 0.5, 0.6 mmol/L in DMEM/F12 medium）. The expression of important genes related to milk protein synthesis process included the genes of the JAK2--STAT5 pathway and the mTOR signaling pathway. All experimentS repeated 3 times. The expression of genes and caseins content were detected by RT--qPCR method and Casein ELISA Kit, respectively. Data were analyzed with the PROC GLM and PROC ANOVA. The results showed that when the concentration of lysine was 1.2 mmol//L and the concentration of methionine was 0.4 mmol/L, the content of casein in medium increased significantly and peaked at 2.95 ppm. We measured the genes expression at this ratio （3：1）.CSN1S1 and LALBA were the most highly expressed genes encoding caseins and increased more than 2--fold,followed by CSN2, CSN3 and CSN2S2, and these genes expression were also up-regulated. The expression of genes related to JAK2--STAT5 pathway were up-- regulated, and JAK2 and ELF5 expression increased significantly. The expression of genes related to roTOR signaling pathway was increased, but S6K was not significantly. EIF4E--BPI expression was significantly down--regulated. The expression of amino acid transporter CAT--1 and ASCT-2 had no change.