【摘要】：乳蛋白的合成需要有效的氨基酸和大量的能量。體內(nèi)實驗證明賴氨酸,蛋氨酸和葡萄糖可以調(diào)節(jié)泌乳奶牛乳蛋白的產(chǎn)量。不僅如此,賴氨酸和蛋氨酸的比例及絕對含量通過細胞信號轉(zhuǎn)導通路JAK2-STAT5和m TOR調(diào)節(jié)乳蛋白合成。本試驗采用體外乳腺上皮細胞培養(yǎng)模型,研究賴氨酸蛋氨酸配比模式和葡萄糖水平對乳蛋白合成的影響,主要關(guān)注對蛋白質(zhì)酪氨酸激酶2(Janus Kinases 2,JAK2)-信號轉(zhuǎn)換和轉(zhuǎn)錄激活因子5(signal transducers and activators of transcription 5,STAT5)和哺乳動物雷帕霉素標靶(mammalian target of rapamycin,m TOR)信號通路的調(diào)節(jié)。原代奶牛乳腺上皮細胞來源于荷斯坦奶牛乳腺組織培養(yǎng),細胞培養(yǎng)在含10%胎牛血清的DMEM/F12培養(yǎng)基中。本試驗為2×2因子設(shè)計,其中兩因素分別為賴氨酸蛋氨酸配比(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)。試驗重復三次。研究包括三方面:試驗一采用MTT法研究不同賴氨酸蛋氨酸配比模式和葡萄糖水平對奶牛乳腺上皮細胞增殖的影響。結(jié)果表明:與低糖處理組相比,高糖處理組的OD值顯著提高(P0.05),即高糖處理組對乳腺上皮細胞的增殖效果顯著高于低糖處理組。HB和HU組相比沒有顯著差異(P0.05),LB和LU組相比沒有顯著差異(P0.05)。賴氨酸蛋氨酸配比模式與葡萄糖水平之間對乳腺上皮細胞增殖沒有交互作用的影響。試驗二采用ELISA法研究不同賴氨酸蛋氨酸配比模式和葡萄糖水平對奶牛乳腺上皮細胞酪蛋白含量的影響。結(jié)果表明:不同賴氨酸蛋氨酸配比模式和葡萄糖水平對奶牛乳腺上皮細胞酪蛋白的合成具有顯著的影響(P0.01)。HB組酪蛋白的合成量最高,HU、LB、LU各組的酪蛋白合成量依次降低。與賴氨酸蛋氨酸失衡組相比,賴氨酸蛋氨酸平衡組顯著提高了酪蛋白的表達水平(P0.01)。與低糖組相比,高糖組顯著提高了酪蛋白的表達水平(P0.01)。試驗三采用Real Time-PCR法研究不同賴氨酸蛋氨酸配比模式和葡萄糖水平對酪蛋白合成關(guān)鍵基因表達的影響。結(jié)果表明:不同賴氨酸蛋氨酸配比模式和葡萄糖水平對奶牛乳腺上皮細胞酪蛋白合成的關(guān)鍵基因有顯著的影響(P0.05)。與賴氨酸蛋氨酸失衡組相比,賴氨酸蛋氨酸平衡組顯著上調(diào)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結(jié)構(gòu)域轉(zhuǎn)錄因子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)基因的表達,同時下調(diào)真核翻譯起始因子4E結(jié)合蛋白1(Eukaryotic translation initiation factor 4E binding protein 1,EIF4E-BP1)基因的表達(P0.05)。與低糖組相比,葡萄糖水平提高顯著上調(diào)了CSN1S2,CSN2,LALBA,STAT5,ELF5,m TOR(P0.01)和CSN1S1(P0.05)基因的表達。賴氨酸蛋氨酸配比模式與葡萄糖水平之間存在交互作用的影響,但是,賴氨酸蛋氨酸配比模式對酪蛋白基因及其合成關(guān)鍵信號通路上的相關(guān)基因的表達更敏感,葡萄糖水平對基因表達的調(diào)節(jié)較弱。綜上所述,提高葡萄糖水平與賴氨酸蛋氨酸配比平衡的組合方式可能直接通過促進乳腺上皮細胞增殖和調(diào)節(jié)酪蛋白合成轉(zhuǎn)錄水平相關(guān)基因表達,從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.
【學位授予單位】：中國農(nóng)業(yè)科學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S816

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