赖氨酸蛋氨酸配比模式和葡萄糖水平影响酪蛋白合成关键基因表达
发布时间：2018-08-24 17:51
【摘要】：乳蛋白的合成需要有效的氨基酸和大量的能量。体内实验证明赖氨酸,蛋氨酸和葡萄糖可以调节泌乳奶牛乳蛋白的产量。不仅如此,赖氨酸和蛋氨酸的比例及绝对含量通过细胞信号转导通路JAK2-STAT5和m TOR调节乳蛋白合成。本试验采用体外乳腺上皮细胞培养模型,研究赖氨酸蛋氨酸配比模式和葡萄糖水平对乳蛋白合成的影响,主要关注对蛋白质酪氨酸激酶2(Janus Kinases 2,JAK2)-信号转换和转录激活因子5(signal transducers and activators of transcription 5,STAT5)和哺乳动物雷帕霉素标靶(mammalian target of rapamycin,m TOR)信号通路的调节。原代奶牛乳腺上皮细胞来源于荷斯坦奶牛乳腺组织培养,细胞培养在含10%胎牛血清的DMEM/F12培养基中。本试验为2×2因子设计,其中两因素分别为赖氨酸蛋氨酸配比(3㑳1和2.3㑳1,即平衡和非平衡)和葡萄糖水平(17.5 mmol/L和2.5 mmol/L,即高糖和低糖)。试验重复三次。研究包括三方面:试验一采用MTT法研究不同赖氨酸蛋氨酸配比模式和葡萄糖水平对奶牛乳腺上皮细胞增殖的影响。结果表明:与低糖处理组相比,高糖处理组的OD值显著提高(P0.05),即高糖处理组对乳腺上皮细胞的增殖效果显著高于低糖处理组。HB和HU组相比没有显著差异(P0.05),LB和LU组相比没有显著差异(P0.05)。赖氨酸蛋氨酸配比模式与葡萄糖水平之间对乳腺上皮细胞增殖没有交互作用的影响。试验二采用ELISA法研究不同赖氨酸蛋氨酸配比模式和葡萄糖水平对奶牛乳腺上皮细胞酪蛋白含量的影响。结果表明:不同赖氨酸蛋氨酸配比模式和葡萄糖水平对奶牛乳腺上皮细胞酪蛋白的合成具有显著的影响(P0.01)。HB组酪蛋白的合成量最高,HU、LB、LU各组的酪蛋白合成量依次降低。与赖氨酸蛋氨酸失衡组相比,赖氨酸蛋氨酸平衡组显著提高了酪蛋白的表达水平(P0.01)。与低糖组相比,高糖组显著提高了酪蛋白的表达水平(P0.01)。试验三采用Real Time-PCR法研究不同赖氨酸蛋氨酸配比模式和葡萄糖水平对酪蛋白合成关键基因表达的影响。结果表明:不同赖氨酸蛋氨酸配比模式和葡萄糖水平对奶牛乳腺上皮细胞酪蛋白合成的关键基因有显著的影响(P0.05)。与赖氨酸蛋氨酸失衡组相比,赖氨酸蛋氨酸平衡组显著上调了αs2-酪蛋白(αs2-casein,CSN1S2),β-酪蛋白(β-casein,CSN2),α-乳清蛋白(α-lactalbumin,LALBA),JAK2,STAT5,ets结构域转录因子5(ets domain transcription factor,ELF5),m TOR(P0.01)和αs1-酪蛋白(αs1-casein,CSN1S1),κ-酪蛋白(κ-casein,CSN3)(P0.05)基因的表达,同时下调真核翻译起始因子4E结合蛋白1(Eukaryotic translation initiation factor 4E binding protein 1,EIF4E-BP1)基因的表达(P0.05)。与低糖组相比,葡萄糖水平提高显著上调了CSN1S2,CSN2,LALBA,STAT5,ELF5,m TOR(P0.01)和CSN1S1(P0.05)基因的表达。赖氨酸蛋氨酸配比模式与葡萄糖水平之间存在交互作用的影响,但是,赖氨酸蛋氨酸配比模式对酪蛋白基因及其合成关键信号通路上的相关基因的表达更敏感,葡萄糖水平对基因表达的调节较弱。综上所述,提高葡萄糖水平与赖氨酸蛋氨酸配比平衡的组合方式可能直接通过促进乳腺上皮细胞增殖和调节酪蛋白合成转录水平相关基因表达,从而提高乳蛋白的合成。
[Abstract]:Lactoprotein synthesis requires effective amino acids and a large amount of energy. In vivo experiments have shown that lysine, methionine and glucose can regulate milk protein production in lactating dairy cows. Moreover, the ratio and absolute content of lysine and methionine regulate milk protein synthesis through JAK2-STAT5 and m TOR signal transduction pathways. External mammary epithelial cell culture model was used to study the effects of lysine-methionine ratio and glucose levels on milk protein synthesis. The main concerns were protein tyrosine kinase 2 (JAK2) - signal transducers and activators of transcription 5 (STAT5) and mammalian rapamycin. The primary dairy mammalian epithelial cells derived from Holstein cow mammary gland tissue culture were cultured in DMEM/F12 medium containing 10% fetal bovine serum. The experiment was designed for 2 *2 factors, in which the ratio of lysine to methionine (3?1 and 2.3?1, i.e. balance) was determined. Non-equilibrium) and glucose levels (17.5 mmol/L and 2.5 mmol/L, high glucose and low glucose). The experiment was repeated three times. The study included three aspects. The first experiment used MTT method to study the effects of different lysine-methionine ratio patterns and glucose levels on the proliferation of dairy cow mammary epithelial cells. There was no significant difference between HB and HU groups (P 0.05), but there was no significant difference between LB and LU groups (P 0.05). There was no interaction between Lysine-methionine ratio and glucose level. The results showed that different lysine-methionine ratios and glucose levels had significant effects on casein synthesis in dairy cow mammary epithelial cells (P 0.01). Compared with the lysine-methionine imbalance group, the lysine-methionine balance group significantly increased the expression level of casein (P 0.01). Compared with the low-glucose group, the high-glucose group significantly increased the expression level of casein (P 0.01). Experiment 3 Real Time-PCR was used to study the different lysine-methionine groups. The results showed that different lysine-methionine ratio patterns and glucose levels had significant effects on the expression of key genes for casein synthesis in dairy cow mammary epithelial cells (P 0.05). Compared with lysine-methionine imbalance group, lysine-methionine balance group had significant effects on the expression of key genes for casein synthesis in dairy cow mammary epithelial cells. Upregulated the expression of alphas2-casein (CSN1S2), beta-casein (CSN2), alpha-whey protein (LALBA), JAK2, STAT5, ETS domain transcription factor 5 (ELF5), m TOR (P 0.01) and alphas1-casein (alphas1-casein, CSN1), kappa-casein (CSN3) (P 0.05). Eukaryotic translation initiation factor 4E binding protein 1 (EIF4E-BP1) gene expression was regulated (P 0.05). Compared with low glucose group, glucose level significantly increased the expression of CSN1S2, CSN2, LALBA, STAT5, ELF5, m TOR (P 0.01) and CSN1S1 (P 0.05). Glucose levels interact, but lysine-methionine ratio patterns are more sensitive to the expression of casein genes and related genes in key signaling pathways for their synthesis, and glucose levels are less sensitive to the regulation of gene expression. It may directly promote the proliferation of mammary epithelial cells and regulate the expression of genes related to casein biosynthesis.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816
[Abstract]:Lactoprotein synthesis requires effective amino acids and a large amount of energy. In vivo experiments have shown that lysine, methionine and glucose can regulate milk protein production in lactating dairy cows. Moreover, the ratio and absolute content of lysine and methionine regulate milk protein synthesis through JAK2-STAT5 and m TOR signal transduction pathways. External mammary epithelial cell culture model was used to study the effects of lysine-methionine ratio and glucose levels on milk protein synthesis. The main concerns were protein tyrosine kinase 2 (JAK2) - signal transducers and activators of transcription 5 (STAT5) and mammalian rapamycin. The primary dairy mammalian epithelial cells derived from Holstein cow mammary gland tissue culture were cultured in DMEM/F12 medium containing 10% fetal bovine serum. The experiment was designed for 2 *2 factors, in which the ratio of lysine to methionine (3?1 and 2.3?1, i.e. balance) was determined. Non-equilibrium) and glucose levels (17.5 mmol/L and 2.5 mmol/L, high glucose and low glucose). The experiment was repeated three times. The study included three aspects. The first experiment used MTT method to study the effects of different lysine-methionine ratio patterns and glucose levels on the proliferation of dairy cow mammary epithelial cells. There was no significant difference between HB and HU groups (P 0.05), but there was no significant difference between LB and LU groups (P 0.05). There was no interaction between Lysine-methionine ratio and glucose level. The results showed that different lysine-methionine ratios and glucose levels had significant effects on casein synthesis in dairy cow mammary epithelial cells (P 0.01). Compared with the lysine-methionine imbalance group, the lysine-methionine balance group significantly increased the expression level of casein (P 0.01). Compared with the low-glucose group, the high-glucose group significantly increased the expression level of casein (P 0.01). Experiment 3 Real Time-PCR was used to study the different lysine-methionine groups. The results showed that different lysine-methionine ratio patterns and glucose levels had significant effects on the expression of key genes for casein synthesis in dairy cow mammary epithelial cells (P 0.05). Compared with lysine-methionine imbalance group, lysine-methionine balance group had significant effects on the expression of key genes for casein synthesis in dairy cow mammary epithelial cells. Upregulated the expression of alphas2-casein (CSN1S2), beta-casein (CSN2), alpha-whey protein (LALBA), JAK2, STAT5, ETS domain transcription factor 5 (ELF5), m TOR (P 0.01) and alphas1-casein (alphas1-casein, CSN1), kappa-casein (CSN3) (P 0.05). Eukaryotic translation initiation factor 4E binding protein 1 (EIF4E-BP1) gene expression was regulated (P 0.05). Compared with low glucose group, glucose level significantly increased the expression of CSN1S2, CSN2, LALBA, STAT5, ELF5, m TOR (P 0.01) and CSN1S1 (P 0.05). Glucose levels interact, but lysine-methionine ratio patterns are more sensitive to the expression of casein genes and related genes in key signaling pathways for their synthesis, and glucose levels are less sensitive to the regulation of gene expression. It may directly promote the proliferation of mammary epithelial cells and regulate the expression of genes related to casein biosynthesis.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S816
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