本文選題：奶牛 + 乳腺上皮細(xì)胞�。� 參考：《浙江大學(xué)》2016年博士論文

【摘要】：乳腺上皮細(xì)胞是乳汁合成的主要場所,與乳產(chǎn)量和乳品質(zhì)密切相關(guān)。乳腺上皮細(xì)胞在生理及病理階段,易發(fā)生氧化應(yīng)激。抵御乳腺上皮細(xì)胞的氧化應(yīng)激和增強其抗氧化能力是維持乳腺健康和高效泌乳的有效手段。白藜蘆醇是一種高效的抗氧化劑,大量研究發(fā)現(xiàn)了它的保健和治療功效,然尚無研究將其用于健康的乳腺抗氧化研究。本研究首先采用H2O2刺激MAC-T細(xì)胞構(gòu)建了奶牛乳腺上皮細(xì)胞氧化應(yīng)激模型,評價了白藜蘆醇抵御乳腺上皮細(xì)胞氧化應(yīng)激的效果。然后利用分子生物學(xué)手段探索了白藜蘆醇通過Nrf2-ARE信號通路、MAPK及PI3K/AKT等信號通路對乳腺上皮細(xì)胞抵御氧化應(yīng)激的保護(hù)作用機理。最后通過向哺乳期ICR小鼠日糧中添加低劑量及高劑量的白藜蘆醇檢測白藜蘆醇在哺乳期飼喂的安全性,并明確其對乳腺的氧化還原平衡及對Nrf2-ARE信號通路的調(diào)控作用。主要研究結(jié)果如下：1.白藜蘆醇保護(hù)奶牛乳腺上皮細(xì)胞抵御H2O2誘導(dǎo)的氧化損傷1.1構(gòu)建H2O2誘導(dǎo)的奶牛乳腺上皮細(xì)胞氧化應(yīng)激模型。采用了不同濃度不同時間的H2O2刺激MAC-T檢測細(xì)胞增殖、細(xì)胞死亡率、ROS蓄積情況、細(xì)胞的氧化平衡情況、細(xì)胞內(nèi)線粒體損傷(BAX和BCL2)和內(nèi)質(zhì)網(wǎng)損傷指標(biāo)(GPR78和CHOP)基因的表達(dá)變化,結(jié)果發(fā)現(xiàn)500μM H2O2處理MAC-T細(xì)胞24 h可以引起顯著地細(xì)胞活力損失,導(dǎo)致細(xì)胞凋亡發(fā)生,處理MAC-T細(xì)胞12 h引起顯著地ROS過量產(chǎn)生,并伴隨著總抗氧能力和超氧化物歧化酶活性的下降,500μM H2O2處理導(dǎo)致GPR78和CHOP基因表達(dá)在4h達(dá)到最大,BAX/BCL2最大值則在24h出現(xiàn)。由此確定500μM以上H2O2處理MAC-T細(xì)胞引起細(xì)胞氧化損傷,主要表現(xiàn)為細(xì)胞活力損傷、細(xì)胞死亡加劇、氧化還原平衡被破壞,并伴隨著線粒體及內(nèi)質(zhì)網(wǎng)損傷,并在后續(xù)研究中采用500μM H2O2處理MAC-T細(xì)胞構(gòu)建氧化應(yīng)激損傷模型。1.2明確白藜蘆醇對H2O2誘導(dǎo)的奶牛乳腺上皮細(xì)胞氧化損傷的保護(hù)效果。采用不同濃度白藜蘆醇處理MAC-T細(xì)胞確定MAC-T 24 h內(nèi)能夠耐受的白藜蘆醇的最高濃度是S0μM。接著基于前期構(gòu)建的氧化應(yīng)激模型,采用50μM以下不同濃度的白藜蘆醇或溶劑對照預(yù)處理2h而后加入H2O2或PBS的處理特定時間來檢測細(xì)胞增殖、細(xì)胞死亡率、ROS蓄積情況、細(xì)胞的氧化平衡情況、細(xì)胞內(nèi)線粒體損傷(BAX和BCL2)和內(nèi)質(zhì)網(wǎng)損傷指標(biāo)(GPR78和CHOP)基因的表達(dá)變化。結(jié)果發(fā)現(xiàn),較之H2O2處理,白藜蘆醇預(yù)處理后再加入H2O2的細(xì)胞活力損失、細(xì)胞凋亡率、細(xì)胞內(nèi)的ROS蓄積程度隨白藜蘆醇的濃度的增加而下降。而且白藜蘆醇的添加增加了細(xì)胞的總抗氧化能力。50μM白藜蘆醇削弱了H2O2誘導(dǎo)的線粒體損傷和內(nèi)質(zhì)網(wǎng)損傷。結(jié)果顯示白藜蘆醇可以保護(hù)乳腺上皮細(xì)胞抵御H2O2誘導(dǎo)的氧化損傷2.白藜蘆醇保護(hù)乳腺上皮細(xì)胞抵御H2O2誘導(dǎo)的氧化損傷的作用機制2.1檢測白藜蘆醇及H2O2處理對奶牛乳腺上皮細(xì)胞中Nrf2-ARE-抗氧化酶鏈的作用。首先檢測白藜蘆醇和H2O2單獨/組合處理MAC-T后細(xì)胞內(nèi)的抗氧化關(guān)鍵基因XNRD1、 HO-1、XCT、NQO-1的表達(dá)豐度變化,發(fā)現(xiàn)白藜蘆醇預(yù)處理后無論是否有H2O2刺激,細(xì)胞內(nèi)的抗氧化關(guān)鍵基因表達(dá)在特定的時間內(nèi)均被激活表達(dá),且較之H2O2單獨處理組,白藜蘆醇+H2O2處理的抗氧化基因均在8h顯著增強。采用不同濃度白藜蘆醇處理細(xì)胞后加入H2O2刺激發(fā)現(xiàn)白藜蘆醇對于抗氧化基因的表達(dá)具有劑量依賴性,有報道證實這些基因是Nrf2-ARE信號通路的靶基因。接著我們檢測發(fā)現(xiàn)白藜蘆醇促進(jìn)了H202處理下Nrf2蛋白的核轉(zhuǎn)移程度,并促進(jìn)了細(xì)胞內(nèi)的ARE原件的激活。由此證實白藜蘆醇激活了細(xì)胞內(nèi)的Nrf2-ARE-抗氧化鏈。2.2明確白藜蘆醇依賴Nrf2發(fā)揮奶牛乳腺細(xì)胞保護(hù)作用。通過siRNA沉默Nrf2基因表達(dá)后重新評估白藜蘆醇的保護(hù)效果,研究發(fā)現(xiàn)細(xì)胞內(nèi)的Nrf2-ARE-抗氧化鏈的關(guān)鍵靶基因表達(dá)在H2O2和白藜蘆醇+H2O2處理中均被抑制。而且siRNA沉默Nrf2基因表達(dá)后白藜蘆醇預(yù)處理不能保護(hù)細(xì)胞免受H2O2誘導(dǎo)的細(xì)胞活力損失,甚至siRNA沉默Nrf2基因表達(dá)后H2O2處理導(dǎo)致的內(nèi)質(zhì)網(wǎng)應(yīng)激加劇。由此得出白藜蘆醇是通過Nrf2-ARE信號通路實現(xiàn)保護(hù)乳腺上皮細(xì)胞的作用的。2.3探索白藜蘆醇通過MAPK、PI3K/AKT信號通路對Nrf2-ARE信號通路激活的作用。檢測白藜蘆醇及H2O2單獨/組合處理MAC-T后細(xì)胞內(nèi)AKT、ERK1/2、JNK1/2、p38的磷酸化水平的時間劑量作用,結(jié)果發(fā)現(xiàn)H202單獨處理激活了所有上述信號轉(zhuǎn)導(dǎo)關(guān)鍵蛋白的活化,而白藜蘆醇預(yù)處理則延長了AKT及ERK1/2的磷酸化時間。后期采用抑制劑預(yù)處理抑制細(xì)胞內(nèi)PI3K/AKT、ERK1/2、JNK1/2、p38信號通路的激活,再采用白藜蘆醇及H2O2單獨/組合處理發(fā)現(xiàn)白藜蘆醇可能是通過EKR、PI3K/AKT信號通路激活Nrf2-ARE信號通路實現(xiàn)保護(hù)乳腺上皮細(xì)胞的作用,而p38信號通路則抑制了白藜蘆醇對Nrf2-ARE信號通路的激活作用及細(xì)胞保護(hù)作用的發(fā)揮。3.日糧添加白藜蘆醇對哺乳期小鼠乳腺氧化損傷及生產(chǎn)性能的影響該部分采用單因素多水平試驗探索了日糧中添加白藜蘆醇對哺乳期ICR小鼠乳腺的抗氧化作用,白藜蘆醇的添加劑量為0、0.02%、0.2%。通過母鼠采食量、體重、器官分?jǐn)?shù)、乳腺重量變化發(fā)現(xiàn)白藜蘆醇的添加不會對母鼠這些指標(biāo)造成負(fù)面影響,通過間接法評估乳產(chǎn)量也未發(fā)現(xiàn)白藜蘆醇具有促進(jìn)乳產(chǎn)量的作用。對仔鼠的窩重,日增重及器官發(fā)育分?jǐn)?shù)檢測發(fā)現(xiàn)白藜蘆醇不會對仔鼠以上發(fā)育指標(biāo)產(chǎn)生負(fù)面作用,提示哺乳期食入一定劑量白藜蘆醇是安全的。通過采集母鼠的乳腺、肝臟、腎臟等器官檢測器官總抗氧化能力、脂質(zhì)過氧化物、超氧化物歧化酶酶活力,發(fā)現(xiàn)白藜蘆醇的添加顯著降低了泌乳期ICR小鼠乳腺中的脂質(zhì)過氧化程度,減少氧化損傷,而對肝臟和腎臟則無上述效果。同時對乳腺的抗氧化基因mRNA表達(dá)的檢測發(fā)現(xiàn),0.2%白藜蘆醇添加的小鼠乳腺中SOD2、GPX1、PRX1、TXNRD1的基因表達(dá)顯著上調(diào),而0.02%以上的白藜蘆醇添加小鼠乳腺中TXNRD1、HO1、Nrf2蛋白表達(dá)均顯著上調(diào),提示日糧添加白藜蘆醇可以減少哺乳期小鼠乳腺氧化損傷,促進(jìn)Nrf2-ARE-抗氧化鏈信號通路。綜上所述,本研究成功構(gòu)建了奶牛乳腺上皮細(xì)胞氧化應(yīng)激模型,利用此模型研究了白藜蘆醇在乳腺上皮細(xì)胞中的抗氧化損傷作用及其機制,并采用在體實驗證實了白藜蘆醇的乳腺保護(hù)作用及其對Nrf2-ARE-抗氧化鏈的調(diào)節(jié)作用。其中Nrf2-ARE-抗氧化鏈在乳腺抗氧化應(yīng)激和細(xì)胞存活中發(fā)揮重要作用,白藜蘆醇可能通過PI3K/AKT、ERK通路影響Nrf2-ARE-抗氧化鏈從而調(diào)控乳腺中的氧化還原平衡。本研究也為白藜蘆醇在奶牛乳腺保護(hù)中的應(yīng)用提供研究基礎(chǔ)及提示。
[Abstract]:Breast epithelial cells are the main sites for milk synthesis, closely related to milk production and milk quality. Breast epithelial cells are prone to oxidative stress at the physiological and pathological stages. It is an effective means to protect breast health and enhance milk secretion by resisting oxidative stress in breast epithelial cells and enhancing their antioxidant capacity. Resveratrol is a highly effective method. Antioxidants, a large number of studies have found its health and therapeutic efficacy, but there is no study on its use in healthy breast antioxidation. First, H2O2 stimulated MAC-T cells to construct a model of oxidative stress in mammary epithelial cells of dairy cows and evaluated the effect of resveratrol against oxidative stress in mammary epithelial cells. Biological means explored the protective mechanism of resveratrol through Nrf2-ARE signaling pathway, MAPK and PI3K/AKT signaling pathway to protect breast epithelial cells against oxidative stress. Finally, the safety of resveratrol in lactation period was detected by adding low dose and high dose of resveratrol into the diet of ICR mice, and the safety of resveratrol in lactation period was determined. Redox balance of mammary glands and regulation of Nrf2-ARE signaling pathway. The main results are as follows: 1. resveratrol protects mammary epithelial cells against oxidative damage induced by H2O2, and constructs a H2O2 induced oxidative stress model of mammary epithelial cells induced by dairy cows. H2O2 stimulates MAC-T detection with different concentrations and time. Cell proliferation, cell mortality, ROS accumulation, cell oxidation balance, intracellular mitochondrial damage (BAX and BCL2) and endoplasmic reticulum damage index (GPR78 and CHOP) gene expression changes. The results showed that 500 u M H2O2 treatment MAC-T cells 24 h can cause significant cell vitality loss, lead to cell apoptosis, and treat MAC-T cells 12 h lead. A significant excess of ROS was produced, accompanied by a decrease in the total oxygen resistance and the activity of superoxide dismutase. The 500 mu M H2O2 treatment resulted in the maximum expression of GPR78 and CHOP genes in 4H and the maximum of BAX/BCL2 in 24h. The cell death was aggravated, the redox balance was destroyed, and the mitochondria and endoplasmic reticulum were damaged. In the follow-up study, 500 M H2O2 was used to treat MAC-T cells to construct oxidative stress damage model.1.2 to clarify the protective effect of resveratrol on the oxidative damage of breast epithelial cells induced by H2O2 in dairy cows. Different concentrations of resveratrol were used to treat M. AC-T cells determined that the highest concentration of resveratrol that could be tolerated in MAC-T 24 h was S0 M. and then based on the pre constructed oxidative stress model, using the resveratrol or solvent control under 50 u M concentrations to pretreat 2H and then add H2O2 or PBS to detect cell proliferation, cell mortality, ROS accumulation, and cells. The changes in the oxidative balance, mitochondrial damage (BAX and BCL2) and the expression of endoplasmic reticulum damage index (GPR78 and CHOP) were found. The results showed that compared with the H2O2 treatment, the cell viability loss, the rate of cell apoptosis and the degree of ROS accumulation in the cells decreased with the increase of the concentration of resveratrol after the pretreatment of resveratrol. The addition of resveratrol increased the total antioxidant capacity of the cells.50 mu M resveratrol weakened the mitochondrial damage induced by H2O2 and the endoplasmic reticulum damage. The results showed that resveratrol protects against H2O2 induced oxidative damage in breast epithelial cells and 2. resveratrol protects breast epithelial cells against H2O2 induced oxidative damage. The effect of resveratrol and H2O2 on the antioxidant enzyme chain of Nrf2-ARE- in mammary epithelial cells of dairy cows was detected by 2.1. First, we detected the changes in the expression of XNRD1, HO-1, XCT, NQO-1 in the cells of resveratrol and H2O2 alone / combined with MAC-T, and found whether the resveratrol was pretreated with H2O2, whether or not there was a H2O2 stimulation. The expression of key antioxidative key genes was activated in a specific time, and the antioxidant genes of resveratrol +H2O2 were significantly enhanced in 8h compared with the H2O2 alone treatment group. The expression of resveratrol was dose-dependent on the expression of antioxidation genes after the use of resveratrol treated cells with different concentrations, and the expression of resveratrol was dose-dependent. The reports confirmed that these genes are the target genes of the Nrf2-ARE signaling pathway. Then we found that resveratrol promoted the degree of nuclear transfer of Nrf2 protein under H202 treatment and promoted the activation of the ARE original in the cells. It was confirmed that resveratrol activated the Nrf2-ARE- antioxidant chain.2.2 in cells to make clear that resveratrol relies on Nrf2 to play milk. The protective effect of bovine mammary gland cells was reevaluated by siRNA silencing Nrf2 gene expression. The study found that the key target gene expression of Nrf2-ARE- antioxidant chain in the cells was suppressed in H2O2 and resveratrol +H2O2 treatment. Moreover, the siRNA silent Nrf2 gene was pretreated with resveratrol and the cells could not protect the cells. Free from H2O2 induced cell vitality loss, even after the siRNA silencing of Nrf2 gene expression, the endoplasmic reticulum stress caused by H2O2 treatment is aggravated. Thus, resveratrol is a.2.3 to protect mammary epithelial cells through the Nrf2-ARE signaling pathway to explore the activation of resveratrol via the MAPK, PI3K/AKT signaling pathway to the Nrf2-ARE signaling pathway. Use. Test the time dose effect of resveratrol and H2O2 alone / combination on the phosphorylation level of AKT, ERK1/2, JNK1/2, p38 in cells after MAC-T. The results showed that H202 activated all the activation of all the key signal transduction proteins, while resveratrol pretreated the phosphorylation time of AKT and ERK1/2. Later, the inhibitors were used as inhibitors. Pretreatment inhibited the activation of intracellular PI3K/AKT, ERK1/2, JNK1/2, p38 signaling pathway, and resveratrol and H2O2 alone / combined treatment found that resveratrol may be activated by EKR, PI3K/AKT signaling pathway to activate Nrf2-ARE signaling pathway to protect mammary epithelial cells, while p38 signaling inhibits resveratrol on Nrf2-ARE. The effect of the activation of the number pathway and the effect of cell protection on the oxidative damage and production performance of.3. diet supplemented with resveratrol on mammary gland of breast-feeding mice. This part uses a single factor and multi level test to explore the antioxidant effect of resveratrol on mammary gland of mammary ICR mice in the diet. The amount of resveratrol is 0,0.02% 0.2%., through the feed intake, body weight, organ fraction, and breast weight change, found that resveratrol did not have a negative effect on the female mice. The effect of resveratrol on milk yield was not detected by indirect method. The litter weight, daily weight gain and organ development score of the offspring were found to be found in resveratrol. It would not have a negative effect on the development index of the offspring, suggesting that a certain dose of resveratrol was safe during lactation. The total antioxidant capacity, lipid peroxide and superoxide dismutase activity were detected by collecting the mammary gland, liver, kidney and other organs of the mice, and the addition of resveratrol significantly reduced the ICR in lactation period. The degree of lipid peroxidation in the rat breast reduced oxidative damage and had no effect on the liver and kidney. At the same time, the expression of antioxidant gene mRNA in the mammary gland was detected. 0.2% the gene expression of SOD2, GPX1, PRX1, TXNRD1 in the mammary gland of mice added with resveratrol was significantly up-regulated, while more than 0.02% of resveratrol added TXNRD in the mammary gland of mice. 1, the expression of HO1 and Nrf2 protein was significantly up-regulated, suggesting that the diet supplemented with resveratrol could reduce the oxidative damage of mammary glands in breast-feeding mice and promote the signaling pathway of Nrf2-ARE- antioxidant chain. The effect of antioxidant injury and its mechanism, and the effects of resveratrol on the protection of resveratrol and its regulation on Nrf2-ARE- antioxidant chain are verified in vivo. The antioxidant chain of Nrf2-ARE- plays an important role in the antioxidant stress and cell survival of the breast. The resveratrol may affect the antioxidant activity of Nrf2-ARE- through the PI3K/AKT and ERK pathway. The chain also regulates the redox balance in the mammary gland. This study also provides a research basis and hint for the application of resveratrol in the protection of mammary gland in dairy cows.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S823

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