长期饲喂高精料日粮对奶山羊肝脏蛋白质表达谱及功能影响的研究

发布时间：2018-07-17 01:33

【摘要】：由于缺乏优质牧草,常给泌乳奶牛饲喂高精料日粮以达到高产奶的目的,然而,长期过度饲喂高精料会引起奶牛瘤胃异常发酵,从而影响奶牛健康,最终降低其泌乳性能。肝脏生理机能是机体重要的保护屏障,乳成分前体物也会经肝脏代谢而进行营养的分配与重分配,从而影响免疫与生产性能。本文以泌乳山羊为动物模型,研究长期采食高精料对瘤胃、血液生理指标及乳品质的影响,并采用2D-MALDITOF/TOF、western blot和real-time PCR技术研究肝脏蛋白质谱的变化,以揭示高精料日粮对泌乳山羊肝脏免疫与代谢的影响及机制;并通过体外原代细胞培养实验,探索内源性脂多糖LPS和瘤胃代谢产物对肝细胞代谢的影响。1高精料对奶山羊生产性能的影响选择12只泌乳中期关中奶山羊,随机分成高精料组(精粗比为65:35)和低精料组(精粗比为35:65),试验期12周。每周记录奶产量,检测乳品质指标。试验结束时,采集瘤胃液、血液和肝脏组织等样品。pH计检测瘤胃液pH值;气象色谱法检测挥发性脂肪酸含量;显色基质法检测瘤胃液LPS浓度;全自动血液生化分析仪检测血浆中白蛋白、胆固醇、血糖等生理生化指标。结果表明:高精料日粮显著降低奶山羊瘤胃液pH(P0.01);升高瘤胃液中丙酸(P0.01)、丁酸(P0.01)、异丁酸(P0.05)、戊酸(P0.01)、异戊酸(P0.01)以及总挥发性脂肪酸VFA(P0.05)的浓度;降低乙酸/丙酸的比值(P0.01),显著增加瘤胃液中LPS((P0.01)浓度。与低精料日粮相比,高精料日粮显著增加山羊乳产量(P0.01)、增加乳糖(P0.01)、乳脂(P0.01)、乳蛋白(P0.01)的含量;增加血液白蛋白(P= 0.07)和低密度脂蛋白胆固醇(P0.05)。2高精料对奶山羊肝脏蛋白表达谱及脂代谢的影响与低精料组相比,高精料组山羊肝体比显著升高(P0.05);肝脏CRP mRNA(P0.05)、SAA mRNA(P0.05)、CD14 mRNA(P0.05)表达显著升高,但 TNF-a mRNA表达有下降的趋势(0.05P0.1)。用2-DE/MALDI-TOF技术鉴定肝脏蛋白表达谱,共得到20个蛋白点,这些蛋白参与机体氧化应激、线粒体功能以及叶酸代谢等的调节。采用ELLSA方法检测结果表明,与低精料组山羊相比,高精料组山羊肝脏总抗氧化能力显著降低(P0.01)。Real-time PCR检测结果表明,线粒体基因COX3 mRNA表达有上升趋势(0.05P0.1),ATP8 mRNA表达显著上调(P0.05)。与低精料组山羊比较,高精料组山羊肝脏胆固醇含量显著降低(P0.01)。Real-time PCR 结果表明,ABCA1(P0.01)、ABCG1(P0.01)、LXRa(P0.01)、LXRβ(P0.05)和SREBP2(P0.01)mRNA表达水平显著上升;肝脏ACAA2(P0.05)、ACSS1(0.05P0.1)、FASN(P0.05)、PPARa(P0.05)、SREBP1c(P0.05)、DGAT1(P0.05)和DGAT2(P0.05)mRNA表达水平均显著上调。3 丁酸钠、LPS对山羊肝细胞糖脂代谢的影响选择2.42kg、45日龄波杂小山羊,处死后分离肝细胞,贴壁培养4h后,在生长培养基中培养72 h,无血清培养基饥饿细胞24 h。用5 mmol/L 丁酸纳、100 ng/mL LPS以及5 mmol/L 丁酸纳和100 ng/mL LPS共同处理肝细胞24 h,收集细胞上清及细胞。检测细胞活力,上清液中葡萄糖含量;检测细胞中糖脂代谢相关基因表达与蛋白含量的差异。结果显示,丁酸钠显著升高肝细胞活性(P0.05);丁酸钠处理显著降低上清液中葡萄糖含量(P0.01)。丁酸钠和LPS均显著下调G6Pase mRNA表达(P0.05),丁酸钠下调 PCK2(P0.05)、PKLR(P0.05)、COX1(P0.05)、SCD(P0.01)mRNA 转录。LPS 上调 ND4(0.05P0.1)和 PFKL(0.05P0.1)mRNA 表达。蛋白免疫印迹结果表明,丁酸纳处理增加GPR41蛋白表达(P0.1);丁酸纳处理引起细胞AMPK蛋白表达升高(0.05P0.1);LPS显著增加肝细胞AMPK(P0.05)和 p-AMPK(0.05P0.1)的蛋白含量。以上研究结果表明:长期饲喂高精料的奶山羊在增加乳产量的同时,瘤胃发生酸中毒,肝脏抗氧化能力降低,线粒体氧化磷酸化增强,脂肪酸氧化增强;丁酸纳和LPS处理原代细胞,丁酸钠通过AMPK通路抑制糖异生,LPS通过AMPK通路激活线粒体氧化磷酸化。
[Abstract]:Due to the lack of high quality pasture, high concentrate diet is often fed to lactating cows for the purpose of high milk production. However, long term overfeeding of high concentrate can cause abnormal fermentation in the rumen of dairy cows, which will affect the health of dairy cows and ultimately reduce their lactation performance. In this paper, lactating goats were used as animal models to study the effects of long term diet high concentrate on rumen, blood physiological indexes and milk quality, and the changes of liver protein mass spectrometry were studied by 2D-MALDITOF/TOF, Western blot and real-time PCR techniques to reveal high essence. Effects and mechanisms of diet on liver immunity and metabolism in lactating goats, the effects of endogenous lipopolysaccharide LPS and rumen metabolites on hepatocyte metabolism were investigated by primary culture in vitro. The effects of.1 high concentrate on milk goat production performance were selected in 12 mid-term dairy goats, which were randomly divided into high concentrate group (65:). 35) and low sperm group (35:65), the test period was 12 weeks. Milk production was recorded every week and milk quality was detected. At the end of the experiment, the pH value of tumor gastric juice was detected by.PH, the content of volatile fatty acids was detected by the method of meteorologic chromatography, the concentration of LPS in the gastric juice was detected by the chromogenic matrix method, and the automatic blood biochemical analysis was carried out. The results showed that high concentrate diet significantly reduced pH (P0.01) of milk goats gastric juice, increased propionic acid (P0.01), butyric acid (P0.01), isobutyric acid (P0.05), valerate (P0.01), isovaleric acid (P0.01) and total volatile fatty acid VFA (P0.05) in the tumor gastric juice, and reduced the ratio of acetic acid / propionic acid. Value (P0.01) significantly increased the concentration of LPS ((P0.01) in gastric juice. Compared with low concentrate diet, high concentrate diet increased goat milk yield (P0.01), increased lactose (P0.01), milk fat (P0.01), milk protein (P0.01) content, increased serum albumin (P= 0.07) and low density lipoprotein cholesterol (P0.05).2 high concentrate on the liver protein expression profiles of dairy goats The effect of lipid metabolism on the liver body ratio of the high concentrate group was significantly higher than that in the low concentrate group (P0.05), and the expression of CRP mRNA (P0.05), SAA mRNA (P0.05) and CD14 mRNA (P0.05) in the liver increased significantly, but the expression of TNF-a mRNA was decreased. The protein expression profiles of the liver were identified with 20 protein points and these proteins were obtained. The results of ELLSA method showed that the total antioxidant capacity of the liver in the high concentrate group was significantly lower than that of the low sperm group (P0.01).Real-time PCR detection results showed that the expression of COX3 mRNA in the mitochondria was rising (0.05P0.1) and ATP8 mRNA table. The cholesterol content in the liver of the high concentrate group was significantly lower than that of the low sperm group (P0.05). The results of the liver cholesterol in the high concentrate group were significantly lower (P0.01).Real-time PCR results showed that ABCA1 (P0.01), ABCG1 (P0.01), LXRa (P0.01), LXR beta (P0.05) and PCR were significantly increased. (P0.05), the expression level of DGAT1 (P0.05) and DGAT2 (P0.05) mRNA significantly up-regulated the sodium butyrate and LPS on the glycolipid metabolism of goat liver cells, selected 2.42kg, 45 day old goat, separated from the liver cells after death, and after adherent culture 4h, the 72 h was cultured in the growth medium, and the serum-free medium starvation cells 24 h. was 5 sodium butyric acid, 100 LPS and 5 mmol/L butyrate and 100 ng/mL LPS were used to treat 24 h of liver cells, collect cell supernatant and cell. Detect cell vitality, glucose content in supernatant, detect the difference of gene expression and protein content in cells. The results show that sodium butyrate significantly increases the activity of liver cells (P0.05); sodium butyrate treatment is significantly reduced. The content of glucose (P0.01) in the supernatant (P0.01). Sodium butyrate and LPS decreased the expression of G6Pase mRNA (P0.05), sodium butyrate downregulated PCK2 (P0.05), PKLR (P0.05), COX1 (P0.05), and the egg white immunoblotting showed that butyrate treatment increased the expression of protein. The increased expression of AMPK protein (0.05P0.1) was caused by acid treatment; LPS significantly increased the protein content of AMPK (P0.05) and p-AMPK (0.05P0.1) in the liver cells. The above results showed that the milk goats fed with high sperm for a long time increased the milk yield, and the rumen was acidosis, the liver antioxidant capacity was reduced, the mitochondrial oxidative phosphorylation was enhanced, and the lipid was increased. The oxidation of fatty acid was enhanced. Sodium butyrate and LPS treated primary cells. Sodium butyrate inhibited gluconeogenesis through AMPK pathway. LPS activated mitochondrial oxidative phosphorylation through AMPK pathway.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S827.5

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