亮氨酸或组氨酸通过哺乳动物雷帕霉素靶蛋白信号通路影响奶牛乳腺上皮细胞中酪蛋白的合成

Effects of Leucine and H.istidine on Milk Protein Synthesis via Mammalian Target of Rapamycin Signaling Pathway in the Bovine Mammary Epithelial Cells

本试验以永生化奶牛乳腺上皮细胞系为模型，单一添加不同浓度的亮氨酸或组氨酸，检测酪蛋白和哺乳动物雷帕霉素靶蛋白（ mTOR）信号通路相关基因的表达，旨在探讨亮氨酸和组氨酸通过mTOR信号通路影响酪蛋白合成的机制。以厄尔平衡溶液代替培养基，并设其为阴性对照，单一添加不同浓度的亮氨酸或组氨酸，分别采用噻唑蓝（ MTT）比色法检测永生化奶牛乳腺上皮细胞12和24 h的增殖；运用实时荧光定量PCR（ qRT？PCR）检测4个酪蛋白编码基因和8个mTOR信号通路相关基因mRNA表达量。结果表明：分别添加不同浓度亮氨酸或组氨酸12和24 h，细胞增殖趋势一致；与阴性对照组相比，分别添加0．15～5．40 mmol／L 亮氨酸或0．15～9．60 mmol／L组氨酸，永生化奶牛乳腺上皮细胞的数量均增加。与阴性对照组相比，当分别添加0．45～10．80 mmol／L亮氨酸6 h时，αs1－酪蛋白（ CSN1S1）、αs2－酪蛋白（ CSN1S2）和κ－酪蛋白（CSN3）基因表达均显著上调（P＜0．05）。当添加0．15～4．80 mmol／L 组氨酸6 h 时， CSN1S1、β－酪蛋白（CSN2）和CSN3基因表达均显著上调（P＜0．05）。在试验组中，当亮氨酸浓度为1．35 mmol／L时，mTOR信号通路相关基因mTOR、mTOR调控蛋白（ raptor）、mTOR复合物1中的绑定蛋白（GβL）、信号下游因子真核翻译起始因子4E结合蛋白1（4EBP1）和真核细胞翻译延伸因子2（eEF2）基因表达量最高。而核糖体S6蛋白激酶（S6K1）基因的表达量随着亮氨酸浓度的增加而减少。当添加组氨酸时，下游信号因子4EBP1、eEF2、真核翻译起始因子4E （ EIF4E）和核糖体蛋白S6（ rps6）基因的表达量随着组氨酸浓度的增加而增加，mTOR基因的表达量随着组氨酸浓度的增加而减少。在试验组中，GβL 的表达量在组氨酸的浓度达到4．80 mmol／L时最高；S6K1基因表达量在组氨酸的浓度达到1．20 mmol／L时最高。综上所述，在乳腺上皮细胞中，亮氨酸和组氨酸能通过mTOR信号通路促进酪蛋白合成相关基因的表达。

This study employed the immortalized bovine mammary epithelial cells （ CMECs-H） as a model to clarify the regulatory effects of single supplement of leucine （ Leu） and histidine （ His） on casein synthesis mo-lecular mechanism via mammalian target of rapamycin （ mTOR） signaling pathway. The casein and expression of genes encoding mTOR signaling were detected by added single supplement of different concentrations of Leu and His. The Earle’ s balanced salts solution without amino acids was set as negative control. After added single supplement of different concentrations of Leu and His, CMECs-H proliferation of 12 and 24 h was detected by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide （MTT） colorimetry, and mRNA expres-sion amounts of 4 casein related genes and 8 mTOR signaling pathway related genes were detected by real-time fluorescence quantitative PCR （ qRT-PCR） . The results showed as follows： after the addition of different con-centrations of Leu or His for 12 and 24 h, the trends of cell proliferation were the same. Compared with nega-tive control group, CMECs-H proliferation increased with added of Leu in the range of 0.15 to 5.40 mmol/L and His in the range of 0.15 to 9.60 mmol/L, respectively. Compared with the negative control group, the ex-pression amounts ofαs1-casein （ CSN1S1） ,αs2-casein （ CSN1S2） andκ-casein （ CSN3） genes measured at 6 h with Leu in the range of 0.45 to 10.80 mmol/L were significantly up-regulated （ P〈0.05） , and the expression amounts of CSN1S1, β-casein （ CSN2） and CSN3 genes measured at 6 h with His in the range of 0. 15 to 4.80 mmol/L were significantly up-regulated （ P〈0.05） . The expression amounts of mTOR signaling pathway related genes, which were mTOR, a binding partner of target of rapamycin （ raptor） , binds to the kinase do-main of mTOR, stabilizes protein of raptor （GβL）, eukaryotic initiation factor 4E binding protein （4EBP1） and eukaryotic elongation factor 2 （ eEF2 ） , were highest in the experimental groups with the addition of 1.35 mmol/L Leu, while the expression amount of the ribosomal protein S6 kinase 1 （ S6K1） gene reduced a-long with the increasing of Leu concentration. When adding His to the negative medium, the expression a-mounts of 4EBP1, eEF2, eukaryotic initiation factor 4E （ EIF4E） and ribosomal protein S6 （ rps6） genes in-creased along with the increasing of His concentration, while the expression amount of mTOR gene reduced a-long with the increasing of His concentration. GβL gene acquired its highest expression amount at 4.80 mmol/L and S6K1 gene at 1.20 mmol/L of His during the experimental groups. These results demonstrate that Leu or His supplements can promote the expression of genes related casein via mTOR signaling pathway related genes in mammary epithelial cells.