奶牛酮体水平与抗氧化、leptin等的关系及leptin等对体外牛肝细胞抗氧化酶表达的影响

发布时间：2018-01-21 04:07

本文关键词： 奶牛酮病氧化应激乳清肝细胞　出处：《广西大学》2015年博士论文　论文类型：学位论文

【摘要】：酮病是危害高产奶牛产后健康的最常见疾病之一,研究该病血浆和乳清中酮体水平与lept in. Insulin.氧化／抗氧化的关系对深入了解该病发病机理具有重要意义。本实验测定了乳清抗氧化指标总抗氧化(TAC),丙二醛(MDA),过氧化氢酶(CAT),谷胱甘肽过氧化物酶(GPX),NEFA(NEFA)和Insulin、leptin、Insulin样生长因子等内分泌因子,按照酮体含量区分健康牛和酮病牛,分析了乳清中酮体和抗氧化指标的相关性以及激素的表达量。本研究利用放射免疫分析(Radio Immuno Assay, RIA)和荧光定量PCR技术以及奶牛肝细胞体外原代分离培养,通过添加不同浓度的leptin、Insulin、Insulin样生长因子,检测犊牛肝细胞氧化应激状态相关指标CAT、GPX活性和mRNA表达量、TAC浓度、MDA含量。为了更深入了解奶牛酮病的发病机理,以便为更好地制定防治措施提供理论依据。实验包括实验一：为了探讨荷斯坦奶牛乳汁酮体含量与部分氧化／抗氧化指标水平的相关性,本研究选择广西某奶牛场泌乳早期的荷斯坦奶牛98头,乳清指标检测结果：乳清KET、TAC、MDA、GPX和CAT水平分别为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)组奶牛各项指标(除CAT外)的水平均高于中(乳酮10～19.9 mg/d1,n=47)、低乳酮组(乳酮10 mg/d1,n=33)的水平,各组KET间存在极显著差异,中、低乳酮组的TAC间差异显著。相关性分析发现全部被检牛KET与TAC间(r=0.237)、TAC与MDA间(r=-0.233)存在显著相关性。高乳酮组MDA与TAC(r=-0.586)、MDA与KET(r=-0.482)存在显著负相关(p0.05),对照组(乳酮20mg/dl)TAC与KET间存在极显著正相关(r=0.298,n=80,p0.01)。结论：研究获得了奶牛乳清酮体和部分氧化／抗氧化指标的测定值。高乳酮对奶牛乳清氧化／抗氧化指标间的相关性会产生明显影响,使TAC与KET相关性变弱,使MDA与TAC负相关性显著增强,使KET与MDA由显著的正相关变为显著负相关。实验二：为探讨酮病奶牛产后乳清和血浆的leptin, Insul in,NEFA的水平及其关系,实验选取健康奶牛7头和酮病奶牛7头,自产后第2周开始到7周,每周采集血样和乳样,检测奶牛乳清和血浆的leptin.Insulin和NEFA的水平。结果表明：酮病牛血浆和乳清leptin,Insulin的水平都降低,而NEFA浓度升高。奶牛乳清NEFA、Insulin、leptin水平显著比血浆水平高。血浆和乳清水平间均有显著相关性,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)。结论,发现了酮病奶牛血浆和乳清leptin,Insulin, NEFA的特征,乳清NEFA的检测具有替代血浆检测的可能性。实验三：本研究通过体外培养犊牛新生肝细胞,测定不同浓度的leptin.Insulin和Insulin样生长因子对肝细胞中抗氧化关键酶表达量的影响。在细胞培养中添加添力口leptin(0、2.5、5、10、50禾口100 ng/ml) Insulin(0.5.10.20.50和100 nM)；Insulin样生长因子(0、10、30、50、100和150 ng/ml)培养24 h后,同时在培养液中检测抗氧化的指标(MDA,TAC,GPX,CAT)前后添加leptin、Insulin和IGFI。结果表明CAT、GPx、β-actin的SYBRGreen Ⅰ荧光定量PCR扩增效率均为100%,标准曲线方程和回归系数分别为y=-3.315x+14.72,R2=0.9992；y=-3.228x+8.78,R2=0.9998；y=-0.301x+8.9344,R2=0.9997。荧光定量PCR溶解曲线为单一峰,特异性强,无引物二聚体。结论：高浓度Insulin和Insulin growth factor 1能增加肝细胞氧化酶CAT,GPX的活性和mRNA表达水平。
[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.

【学位授予单位】：广西大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S858.23

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