本文關(guān)鍵詞： 奶牛 酮病 氧化應(yīng)激 乳清 肝細(xì)胞　出處：《廣西大學(xué)》2015年博士論文　論文類型：學(xué)位論文

【摘要】：酮病是危害高產(chǎn)奶牛產(chǎn)后健康的最常見疾病之一,研究該病血漿和乳清中酮體水平與lept in. Insulin.氧化／抗氧化的關(guān)系對(duì)深入了解該病發(fā)病機(jī)理具有重要意義。本實(shí)驗(yàn)測(cè)定了乳清抗氧化指標(biāo)總抗氧化(TAC),丙二醛(MDA),過氧化氫酶(CAT),谷胱甘肽過氧化物酶(GPX),NEFA(NEFA)和Insulin、leptin、Insulin樣生長(zhǎng)因子等內(nèi)分泌因子,按照酮體含量區(qū)分健康牛和酮病牛,分析了乳清中酮體和抗氧化指標(biāo)的相關(guān)性以及激素的表達(dá)量。本研究利用放射免疫分析(Radio Immuno Assay, RIA)和熒光定量PCR技術(shù)以及奶牛肝細(xì)胞體外原代分離培養(yǎng),通過添加不同濃度的leptin、Insulin、Insulin樣生長(zhǎng)因子,檢測(cè)犢牛肝細(xì)胞氧化應(yīng)激狀態(tài)相關(guān)指標(biāo)CAT、GPX活性和mRNA表達(dá)量、TAC濃度、MDA含量。為了更深入了解奶牛酮病的發(fā)病機(jī)理,以便為更好地制定防治措施提供理論依據(jù)。實(shí)驗(yàn)包括實(shí)驗(yàn)一：為了探討荷斯坦奶牛乳汁酮體含量與部分氧化／抗氧化指標(biāo)水平的相關(guān)性,本研究選擇廣西某奶牛場(chǎng)泌乳早期的荷斯坦奶牛98頭,乳清指標(biāo)檢測(cè)結(jié)果：乳清KET、TAC、MDA、GPX和CAT水平分別為13.8±6.7mg/dl.5.0±3.1 U/ml、2.2±1.8 nmol/ml、56.1±47.1 U/ml和3.0±2.0U/ml。高乳酮(乳酮≥20 mg/d1,n=18)組奶牛各項(xiàng)指標(biāo)(除CAT外)的水平均高于中(乳酮10～19.9 mg/d1,n=47)、低乳酮組(乳酮10 mg/d1,n=33)的水平,各組KET間存在極顯著差異,中、低乳酮組的TAC間差異顯著。相關(guān)性分析發(fā)現(xiàn)全部被檢牛KET與TAC間(r=0.237)、TAC與MDA間(r=-0.233)存在顯著相關(guān)性。高乳酮組MDA與TAC(r=-0.586)、MDA與KET(r=-0.482)存在顯著負(fù)相關(guān)(p0.05),對(duì)照組(乳酮20mg/dl)TAC與KET間存在極顯著正相關(guān)(r=0.298,n=80,p0.01)。結(jié)論：研究獲得了奶牛乳清酮體和部分氧化／抗氧化指標(biāo)的測(cè)定值。高乳酮對(duì)奶牛乳清氧化／抗氧化指標(biāo)間的相關(guān)性會(huì)產(chǎn)生明顯影響,使TAC與KET相關(guān)性變?nèi)?使MDA與TAC負(fù)相關(guān)性顯著增強(qiáng),使KET與MDA由顯著的正相關(guān)變?yōu)轱@著負(fù)相關(guān)。實(shí)驗(yàn)二：為探討酮病奶牛產(chǎn)后乳清和血漿的leptin, Insul in,NEFA的水平及其關(guān)系,實(shí)驗(yàn)選取健康奶牛7頭和酮病奶牛7頭,自產(chǎn)后第2周開始到7周,每周采集血樣和乳樣,檢測(cè)奶牛乳清和血漿的leptin.Insulin和NEFA的水平。結(jié)果表明：酮病牛血漿和乳清leptin,Insulin的水平都降低,而NEFA濃度升高。奶牛乳清NEFA、Insulin、leptin水平顯著比血漿水平高。血漿和乳清水平間均有顯著相關(guān)性,NEFA(n=84,r=0.810,P=0.001), lept in(n=84, r=0.64, P=0.025), Insul in(n=84,r=-0.627,P=0.029)。結(jié)論,發(fā)現(xiàn)了酮病奶牛血漿和乳清leptin,Insulin, NEFA的特征,乳清NEFA的檢測(cè)具有替代血漿檢測(cè)的可能性。實(shí)驗(yàn)三：本研究通過體外培養(yǎng)犢牛新生肝細(xì)胞,測(cè)定不同濃度的leptin.Insulin和Insulin樣生長(zhǎng)因子對(duì)肝細(xì)胞中抗氧化關(guān)鍵酶表達(dá)量的影響。在細(xì)胞培養(yǎng)中添加添力口leptin(0、2.5、5、10、50禾口100 ng/ml) Insulin(0.5.10.20.50和100 nM)；Insulin樣生長(zhǎng)因子(0、10、30、50、100和150 ng/ml)培養(yǎng)24 h后,同時(shí)在培養(yǎng)液中檢測(cè)抗氧化的指標(biāo)(MDA,TAC,GPX,CAT)前后添加leptin、Insulin和IGFI。結(jié)果表明CAT、GPx、β-actin的SYBRGreen Ⅰ熒光定量PCR擴(kuò)增效率均為100%,標(biāo)準(zhǔn)曲線方程和回歸系數(shù)分別為y=-3.315x+14.72,R2=0.9992；y=-3.228x+8.78,R2=0.9998；y=-0.301x+8.9344,R2=0.9997。熒光定量PCR溶解曲線為單一峰,特異性強(qiáng),無引物二聚體。結(jié)論：高濃度Insulin和Insulin growth factor 1能增加肝細(xì)胞氧化酶CAT,GPX的活性和mRNA表達(dá)水平。
[Abstract]:Ketosis is one of the most common disease of high-yielding dairy cows postpartum health, disease and study the relationship between plasma whey and lept in. ketone levels Insulin. oxidation and antioxidation has important significance for understanding the pathogenesis of the disease. Whey antioxidant indexes of total antioxidant were determined in this experiment (TAC), malondialdehyde (MDA), hydrogen peroxide the enzyme (CAT), glutathione peroxidase (GPX), NEFA (NEFA) and Insulin, leptin, Insulin like growth factor, endocrine factor, in accordance with the contents of ketone to distinguish healthy cows and ketosis in cattle, analyzed the ketone and antioxidant indices in whey and the correlation between the expression of hormone. Radioimmunoassay was used in this study (Radio Immuno Assay, RIA) and fluorescence quantitative PCR and bovine hepatocytes isolated and cultured in vitro, by adding different concentrations of leptin, Insulin, Insulin like growth factor, oxidation of calf hepatocytes should be detected Relevant indicators of excitation state CAT expression, GPX activity and mRNA concentration of TAC, the content of MDA. In order to better understand the pathogenesis of ketosis, in order to better formulate prevention measures and provide a theoretical basis. The experiment includes Experiment 1: in order to explore the relationship between the Holstein cows and milk ketone content in partial oxidation / antioxidant index level, the study on the selection of Guangxi cows in early lactation Holstein cows 98, the detection results of whey whey KET index: TAC, MDA, GPX, and CAT levels were 13.8 + 6.7mg/dl.5.0 3.1 + U/ml, 2.2 + 1.8 + 47.1 nmol/ml, 56.1 U/ml and 3 + 2.0U/ml. high milk (milk for more than 20 mg/d1, n=18) the cow group indexes (except CAT) levels were higher than those in (milk ketone 10 ~ 19.9 mg/d1, n=47), low milk ketone group (milk ketone 10 mg/d1, n=33) level, there are significant differences between groups KET, the difference between the low milk ketone group TAC significantly. Analysis found that all tested bovine KET and TAC (r=0.237), TAC and MDA (r=-0.233). There was significant correlation between the high milk ketone group MDA and TAC (r=-0.586), MDA and KET (r=-0.482) had a significant negative correlation (P0.05), control group (milk ketone 20mg/dl) has extremely significant positive correlation TAC and KET (r=0.298, n=80, P0.01). Conclusion: the research has obtained the determination of ketone bodies and partial oxidation / cow whey antioxidant index value. High milk will have a significant impact on the relationship between dairy whey oxidation / antioxidant index between TAC and KET, the correlation is weak, the MDA and TAC negative correlation significantly enhanced so, KET and MDA are significantly related to a significant negative correlation. Experiment two: in order to investigate the ketosis in dairy cows postpartum whey and plasma leptin, Insul, in, NEFA level and its relationship, selected 7 cows and healthy dairy cows in 7, produced by the beginning of the 7 week after second weeks of weekly collection blood and The milk samples of dairy whey and detected the level of plasma leptin.Insulin and NEFA. The results showed that ketosis in bovine plasma and whey leptin, Insulin levels were decreased, while NEFA concentration was increased. The cow whey NEFA, Insulin, leptin were significantly high than plasma levels. Plasma and whey levels were significant correlation (n=84, NEFA, r=0.810, P=0.001, lept) in (n=84, r=0.64, P=0.025), Insul in (n=84, r=-0.627, P=0.029). Conclusion, found ketosis plasma and whey leptin, the features of NEFA, Insulin, NEFA has the possibility to replace the detection of whey plasma detection. Experiment three: the study by in vitro culture of newborn calves the amount of liver cells, to determine the effects of different concentrations of leptin.Insulin and Insulin like growth factor on the expression of key enzymes of antioxidant in liver cells. Add Tim leptin in cell culture (0,2.5,5,10,50 and 100 ng/ml Insulin (0.5.10.20 port) .50 and 100 nM); Insulin like growth factor (0,10,30,50100 and 150 ng/ml) after 24 h culture, while culture detection in liquid antioxidant index (MDA, TAC, GPX, CAT) before and after the addition of leptin, Insulin and IGFI. results show that CAT, GPx, SYBRGreen 1 fluorescent quantitative PCR beta -actin amplification efficiency were 100%, the standard curve equation and the regression coefficients were y=-3.315x+14.72, R2=0.9992; y=-3.228x+8.78, R2=0.9998; y=-0.301x+8.9344, R2=0.9997. fluorescence quantitative PCR solubility curve is a single peak, strong specificity, no primer dimer two. Conclusion: high concentration of Insulin and Insulin growth factor 1 can increase the liver enzymes CAT, GPX expression level and activity of mRNA.

【學(xué)位授予單位】：廣西大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：S858.23

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 凌澤繼;楊憲苓;趙海洋;楊賓賓;何寶祥;;奶牛胎衣不下與血漿GPX、CAT活性的相關(guān)研究[J];廣西農(nóng)業(yè)科學(xué);2010年09期

2 武瑞,張洪友,夏成,肖立群,富艷玲;荷斯坦奶牛酮病對(duì)抗氧化系統(tǒng)影響的研究[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2005年01期

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本文編號(hào)：1450417