赖氨酸对奶牛乳腺上皮细胞内乳蛋白合成相关基因表达和蛋白磷酸化的影响

Effects of Lysine on Gene Expressions and Protein Phosphorylation Involved in Milk Protein Synthesis in Bovine Mammary Epithelial Cells

本试验旨在研究赖氨酸(Lys)对奶牛乳腺上皮细胞(BMECs)内乳蛋白合成相关基因表达和蛋白磷酸化的影响,深入探讨Lys对乳蛋白合成影响的机理。将第3代BMECs随机分为6组,各组培养基中Lys的浓度分别为0.5(对照)、1.0、2.0、4.0、8.0和16.0 mmol/L,每组6个重复。37℃、5%CO_2培养48 h,之后采用化学发光法测定BMECs内三磷酸腺苷(ATP)的含量,采用实时荧光定量PCR法测定乳蛋白合成相关基因表达量以及采用蛋白质免疫印迹法测定乳蛋白合成相关蛋白的磷酸化水平。结果表明:随着Lys浓度的增加,ATP含量呈趋于显著的二次曲线升高(P=0.050);κ-酪蛋白(CSN3)(P=0.093)、哺乳动物雷帕霉素靶蛋白(mTOR)(P=0.005)、真核起始因子4E(e IF4E)(P=0.076)和磷酸腺苷活化的蛋白激酶α1(AMPKα1)基因表达量(P=0.045)呈显著或趋于显著的二次曲线变化,均为先升高后降低;α-酪蛋白(CSN1S1)基因表达量(P=0.081)及mTOR(P=0.038)和p70核糖体蛋白S6激酶1(S6K1)磷酸化水平(P=0.022)呈显著或趋于显著的一次线性降低;磷酸腺苷活化的蛋白激酶(AMPK)的磷酸化水平呈显著的一次线性升高(P=0.014)。方差分析结果显示,添加Lys显著影响ATP含量、乳蛋白合成相关基因表达量及e IF4E磷酸化水平(P<0.05),其中,ATP含量以2.0~16.0 mmol/L组,CSN1S1、β-酪蛋白(CSN2)、信号转导和转录激活因子5(STAT5)基因表达量以1.0~2.0 mmol/L组,mTOR基因表达量以1.0~8.0 mmol/L组,CSN3基因表达量以1.0~4.0 mmol/L组,酪氨酸激酶2(JAK2)基因表达量以1.0~16.0 mmol/L组,S6K1基因表达量以2.0 mmol/L组,e IF4E基因表达量以2.0~8.0 mmol/L组,e IF4E磷酸化水平以2.0~4.0 mmol/L组时促进效果较好,但16.0 mmol/L组CSN1S1、CSN3、STAT5及mTOR基因表达量受到抑制,1.0~16.0 mmol/L组真核起始因子4E结合蛋白1(4EBP1)基因表达量受到抑制。总之,Lys浓度为1.0~2.0 mmol/L时,对BMECs内乳蛋白合成相关基因表达的促进效果较好。

The objective of this study was to determine the effects of lysine（ Lys） on gene expressions and protein phosphorylation involved in milk protein synthesis in bovine mammary epithelial cells（ BMECs）. The3 rd generation BMECs were randomly divided into six group with six replicates per group,cells in different groups were cultured in medium with 0.5（ control）,1.0,2.0,4.0,8.0 and 16.0 mmol/L Lys,respectively.After 48 h cultivation at 37 ℃ and 5% CO_2,adenosine triphosphate（ ATP） content was measured by the method of chemiluminescence,expression levels of genes involved in milk protein synthesis were determined by real-time PCR,and phosphorylation levels of proteins involved in milk protein synthesis were determined by Western blotting method. The results showed as follows： with the increase of Lys concentration,ATP content tended to be quadratically significantly increased（ P = 0. 050）; expression levels of κ-casein（ CSN3）（ P =0.093）,mammalian target of rapamycin（ mTOR）（ P = 0.005）,eukaryotic initiation factor 4 E（ e IF4 E）（ P =0.076） and adenosine 5＇-mono-phpsphate-active protein kinase α1（ AMPKα1） genes（ P = 0. 045） were significantly or tended to significantly quadratically changed,all showed firstly increased then decreased; expression level of αs1-casein（ CSN1 S1） gene（ P = 0.081）,phosphorylation levels of mTOR（ P = 0.038） and ribosomal protein S6 kinase 1（ S6 K1）（ P = 0. 022） were significantly or tend to significantly linearly decreased;phosphorylation level of adenosine 5＇-mono-phpsphate-active protein kinase（ AMPK） was significantly linearly increased（ P = 0.014）. The results of analysis of variance showed that the supplementation of Lys had significantly effects on ATP content,expression levels of genes involved in milk protein synthesis and phosphorylation level of e IF4 E（ P 〈 0. 05）,among them,the improvement effects of 2. 0 to 16. 0 mmol/L Lys for ATP content,1.0 to 2.0 mmol/L Lys for CSN1 S1,β-casein（ CSN2）,signal transducer and activator of transcription 5（ STAT5） gene expression levels,1. 0 to 8. 0 mmol/L Lys for mTOR gene expression level,1. 0 to4.0 mmol/L Lys for CSN3 gene expression level,1.0 to 16. 0 mmol/L Lys for Janus kinase 2（ JAK2） gene expression level,2.0 mmol/L Lys for S6 K1 gene expression level,2.0 to 8.0 mmol/L Lys for e IF4 E gene expression level,2.0 to 4.0 mmol/L Lys for e IF4 E phosphorylation level were better,however,16. 0 mmol/L Lys had inhibition effects on expression levels of CSN1 S1,CSN3,STAT5 and mTOR genes,and 1. 0 to16.0 mmol/L Lys had inhibition effect on expression level of eukaryotic initiation 4 E binding protein 1（4 EBP1） gene. In conclusion,the optimal Lys concentration for gene expressions involved in milk protein synthesis in BMECs is 1.0 to 2.0 mmol/L.