本文關鍵詞： MAC-T 單一EAA 信號蛋白 泌乳小鼠 仔鼠體增重　出處：《山東農業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：近年來的研究結果表明,氨基酸不僅是蛋白質合成的底物,而且能夠作為生物信號直接參與蛋白質合成的調節(jié)。為研究必需氨基酸(EAA)通過細胞內信號途徑調控乳蛋白合成的機制,本文以牛乳腺上皮細胞系(MAC-T)為材料,系統(tǒng)研究了10種EAA對調控乳蛋白合成關鍵信號途徑的影響,以泌乳小鼠為模型,以仔鼠體增重衡量蛋白合成速率研究了調控乳蛋白合成潛力EAA對泌乳的影響及機制。單一EAA對調控乳蛋白合成關鍵信號蛋白磷酸化程度的影響。本試驗以MAC-T為試驗材料,試驗處理為在依次缺失一種EAA的DMEM/F12培養(yǎng)基上培養(yǎng),以含有全部EAA的DMEM/F12培養(yǎng)基培養(yǎng)為正對照,以缺失全部EAA的DMEM/F12培養(yǎng)基培養(yǎng)為負對照。分別缺失10種EAA對蛋白合成過程中主要信號蛋白磷酸化程度有極顯著影響,負對照mTOR磷酸化程度下降58%,缺失Met、Ile或Val使mTOR磷酸化程度分別下降38%、41%、42%;單一EAA缺失降低細胞內4eBP1的磷酸化程度,其中缺失Met、Ile、Thr和Lys分別下降39%、41%、36%和42%;缺失Met、Ile、Leu、Val時S6K1的磷酸化程度分別下降33%、38%、41%、40%;單一EAA缺失顯著提高eEF2磷酸化程度,其中缺失Met、Lle、Val磷酸化程度分別提高18%、20%、18%;缺失所有EAA和Met eIF2α磷酸化程度分別提高49%和39%。結果表明,單一EAA主要影響細胞內mTOR和GCN2 2條通路中關鍵信號蛋白的磷酸化,且關鍵信號蛋白對完全缺失Met、Leu、Ile、Thr、Val、Lys反應敏感。Met、Leu、Ile、Val對調控乳蛋白合成關鍵信號蛋白的影響。為研究信號蛋白反應敏感EAA的作用效果,以MAC-T為試驗材料,首先研究不同濃度EAA對調控蛋白合成關鍵信號蛋白的影響以確定細胞內EAA缺乏狀態(tài),EAA缺乏時添加Met、Leu、Ile、Val以及這四種EAA兩兩組合。隨著EAA濃度的變化信號蛋白磷酸化程度符合米氏方程變化趨勢,EAA濃度20%以下信號蛋白磷酸化程度變化幅度較大,當EAA濃度在30%-90%之間信號蛋白的磷酸化程度變化趨于穩(wěn)定,據(jù)此確定10%為后續(xù)研究EAA缺乏狀態(tài)的濃度。EAA缺乏狀態(tài)添加Met、Leu、Ile、Val以及這四種EAA兩兩組合,添加Leu、Ile、Met和Leu、Met和Ile、Leu和Ile mTOR磷酸化程度分別提高47%、69%、187%,200%和165%;單獨添加Leu、Ile、Val提高4eBP1的磷酸化程度,且同時添加Met和Leu、Leu和Ile、Val和Ile有加性效果;添加Ile S6K1磷酸化程度提高63%,且Ile與其他三種氨基酸組合有加性作用。結果表明,EAA缺乏時添加Met、Leu、Ile、Val影響提高mTOR通路中信號蛋白的磷酸化程度,且四種氨基酸兩兩組合對信號蛋白的磷酸化有疊加作用。Leu、Ile、Met、Thr對泌乳小鼠乳蛋白合成的影響。為評估體外試驗中乳蛋白合成調控潛力EAA對泌乳的貢獻及作用機制,首先確定小鼠蛋白缺乏狀態(tài),建立仔鼠體增重和不同蛋白水平的反應曲線,試驗選取分娩日期相近的孕鼠,日糧蛋白水平分別為6%、9%、12%、15%、18%、21%、24%,和27%。當?shù)鞍姿降陀?1%時,隨蛋白濃度的增加仔鼠體增重增加,仔鼠體增重隨蛋白水平的提高成米氏反應趨勢,最大體增重一半時的蛋白水平為14%,最大體增重為113g;隨日糧蛋白水平提高泌乳小鼠乳腺組織中S6K1磷酸化程度提高,eEF2的磷酸化程度降低。根據(jù)以上結果,確定15%為日糧低蛋白,即負對照,21%為日糧高蛋白,即正對照,低蛋白基礎上分別添加Met、Leu、Ile、Thr。低蛋白日糧添加Met、Leu、Ile仔鼠體增重分別提高10%、11%、9%;添加Met、Leu、Ile和Thr后泌乳小鼠乳腺組織中mTOR的磷酸化程度分別提高了55%、34%,、47%和45%;必需氨基酸處理提高泌乳小鼠血漿IGF-1的濃度;仔鼠窩增重和乳腺組織中mTOR,相關系數(shù)為0.40。結果表明,低蛋白日糧添加Met、Leu、Ile獨立刺激乳蛋白合成速率(仔鼠窩增重),且主要通過mTOR信號通路。Met、Leu誘導內分泌變化對泌乳的影響。初探泌乳潛力EAA(Met、Leu)誘導內分泌的變化對泌乳的影響機制,本試驗以泌乳小鼠為模型,以仔鼠體增重衡量蛋白合成速率,低蛋白日糧添加Met、Leu。Met、Leu提高泌乳小鼠仔鼠體增重;添加Met提高泌乳小鼠血漿胰島素水平,Met、Leu提高血漿中IGF-1水平,提高乳腺組織中Akt的磷酸化程度;Met明顯增加泌乳小鼠胰腺中胰島素分泌和肝臟中IGF-1的分泌,且Met、Leu引起的肝臟差異基因主要富集在胰腺分泌和胰島素信號途徑。綜上,在體外,單一EAA主要影響細胞內mTOR和GCN2通路中關鍵信號蛋白的磷酸化,Met、Leu、Ile、Val對細胞內控制蛋白合成關鍵信號蛋白的磷酸化有單獨和疊加作用;在體內,蛋白缺乏日糧補充Met、Leu、Ile獨立刺激乳蛋白合成速率(仔鼠窩增重),可見“木桶理論”不適用于泌乳動物,且Met、Leu、Ile對乳蛋白合成速率的影響主要通過mTOR信號通路,不依賴于GCN2信號通路,Met、Leu誘導激素的變化是EAA調控乳蛋白合成的另一重要途徑。
[Abstract]:Recent results show that not only is the amino acid substrates for protein synthesis, and can be used as a biological signal directly involved in the regulation of protein synthesis. For the study of essential amino acids (EAA) through the mechanism of intracellular signal pathway for the synthesis of the regulation of milk protein, milk in the gland epithelial cell line (MAC-T) as material, studied 10 EAA on the regulation of milk protein synthesis of key signaling pathways, in lactating mice with the body weight gain of offspring to measure the rate of protein synthesis on the regulation of milk protein synthesis and potential effects of EAA on lactation mechanism. The effect of single EAA on the regulation of milk protein synthesis key signal protein phosphorylation. This experiment based on MAC-T the test material, test for processing medium in order to loss of a EAA DMEM/F12, which contains all the EAA DMEM/F12 medium as positive control, with the lack of full EAA DMEM/F 12 medium as negative control. Respectively. 10 EAA had significant effect on protein synthesis in the process of the main signal protein phosphorylation, negative control mTOR phosphorylation decreased 58%, deletion of Met, Ile or Val to mTOR phosphorylation decreased 38%, respectively, 41%, 42%; single EAA deficiency reduced intracellular the phosphorylation of 4eBP1, including Ile, Thr and loss of Met and Lys were decreased by 39%, 41%, 36% and 42%; Ile, Leu, Met deletion, Val phosphorylation of S6K1 were decreased by 33%, 38%, 41%, 40%; single EAA deletion significantly increased eEF2 phosphorylation level in the absence of Met. Lle, phosphorylation of Val were increased by 18%, 20%, 18%; all EAA and Met lack of eIF2 alpha phosphorylation were increased by 49% and 39%. results showed that EAA mainly affects the key single intracellular signal protein mTOR and GCN2 2 pathways of phosphorylation and protein of key signal complete absence of Met, Leu. Ile, Thr, Val, Lys Sensitive.Met, Leu, Ile, Val impact on the regulation of milk protein synthesis of key signaling proteins. For the effect of signal protein was sensitive to EAA, with MAC-T as test material, first study the effects of different concentrations of EAA on regulation of protein synthesis key signaling proteins to determine intracellular EAA deficiency, add Met, EAA the lack of Leu, Ile, Val and the four EAA 22. With the combination of signal protein phosphorylation of EAA concentration changes consistent with Michaelis equation change trend, EAA concentration below 20% signal protein phosphorylation changes greatly, when the concentration of EAA in 30%-90% phosphorylation signal protein changes tended to be stable, to determine the 10% for further study of EAA lack of concentration.EAA state condition of lack of adding Met, Leu, Ile, Val and the four EAA 22, adding Leu, Ile, Met and Leu, Met and Ile, Leu and Ile phosphorylation of mTOR were increased 47%, 69%, 187%, 200% and 165%; Ile Val, adding Leu alone, increased the phosphorylation of 4eBP1, and the addition of Met and Leu, Leu and Ile, Val and Ile had additive effect; adding Ile S6K1 phosphorylation increased 63%, and the Ile and other three kinds of amino acids in combination with additive effects. The results indicate that the addition of Met, EAA Leu Ile, Val deficiency, affecting the increase of signal protein in mTOR pathway phosphorylation, phosphorylation of 22 amino acids and four kinds of combination of signaling proteins have additional effects on.Leu, Ile, Met, Thr on the synthesis of lactation mice milk protein. For contribution and mechanism in vitro evaluation of milk protein synthesis control potential of EAA on lactation, first determine the mouse protein deficiency, establish rat body weight and increase the reaction curve of different protein levels, were selected to the date of delivery is similar in pregnant rats. Dietary protein levels were 6%, 9%, 12%, 15%, 18%, 21%, 24% when the eggs, and 27%. The white level is less than 21%, with the body weight gain of offspring increased protein concentration increased, the body weight gain of rats with increased levels of protein into the Michaelis Menten reaction trend, most generally increase protein levels for the 14% half of weight, the gross weight of 113g; S6K1 phosphorylation level of lactating mammary gland in mice with increased the level of dietary protein, phosphorylation of eEF2 decreased. According to the above results, determine the 15% low dietary protein, negative control, 21% for high protein diets, positive control, low protein basis were added to Met, Leu, Ile, Thr. low protein diets adding Met, Leu, weight increased 10%, Ile rats increase 11%, 9%; Leu, add Met, mTOR Ile and Thr lactating mouse mammary gland after phosphorylation were increased by 55%, 34%, 47%, and 45%; essential amino acid treatment increased the concentration of IGF-1 in plasma of lactating rats; nest mTOR weight gain and mammary gland tissue, the correlation coefficient is 0.40.. The results show that low protein diets containing Met, Leu, Ile independent stimulation of milk protein synthesis rate (rat nest weight), and mainly through the mTOR.Met pathway and effects of Leu induced endocrine changes on lactation of lactation. The potential of EAA (Met, Leu) changes induced by endocrine influence mechanism on lactation, the in order to test lactating mice with the body weight gain of offspring to measure the rate of protein synthesis, low protein diet supplemented with Met, Leu.Met, Leu increase the milk of mice body weight gain; improve the level of insulin, mouse plasma Met Met Leu add milk, improve the level of IGF-1 in plasma, improve the breast tissue of the phosphorylation of Akt Met; increased secretion of pancreatic insulin secretion in lactating mice and IGF-1 in liver and Met, Leu induced differences in gene expression of liver mainly enriched in pancreatic secretion and insulin signaling pathway. In conclusion, the main effect of EAA in vitro, single cell mTOR and GCN2 The key signal pathway in protein phosphorylation, Met, Leu, Ile, Val control protein synthesis key signaling proteins to intracellular phosphorylation alone and superposition; in vivo, lack of protein diets supplemented with Met, Leu, Ile independent stimulation of milk protein synthesis rate (rat nest weight), visible "cask the theory does not apply to lactating animal, and Met, Leu, Ile influence on milk protein synthesis rate mainly through the mTOR pathway, does not depend on the GCN2 signaling pathway, Met, Leu induced changes in hormone is another important way to EAA regulation of milk protein synthesis.

【學位授予單位】：山東農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S852.2

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