能量负平衡奶牛产后卵巢静止血清蛋白质组学分析及其预警评估
发布时间：2018-09-06 07:50
【摘要】：当前,卵巢静止已成为集约化牛场高产奶牛产后常见的卵泡生长异常的疾病。其特征为卵巢或相关的卵巢结构出现无周期性特征的静止状态。奶牛产后卵巢上卵泡生长受到诸多因素的影响。尽管能量负平衡被公认为奶牛产后卵巢静止的主要病因,但是奶牛产后发生卵巢静止时,机体整体蛋白表达的情况,以及与该病的关系还未见报道。为此,本试验应用蛋白组学中荧光差异双向凝胶电泳(Fluorescence Two-dimensional Differential Gel Electrophoresis,2D-DIGE)与基质辅助激光解析离子化飞行时间质谱(MatrixAssisted Laser Desorption/Lonization Time of Flight Mass Spectrometry,MALDI-TOF-MS)联用技术,结合生物信息学分析,获得了能量负平衡所致的卵巢静止奶牛血清差异表达蛋白,探究了这些差异表达蛋白与卵巢静止之间的关系,为今后深入探索奶牛产后卵巢静止发生机制提供科学依据。本试验在黑龙江某集约化奶牛场选取了年龄、胎次相近的经产奶牛,在产后14~21d根据血清中β-羟丁酸(β-hydroxybutyrate,BHBA)、游离脂肪酸(Non esterified fatty acid,NEFA)和葡萄糖(Glucose,Glc)含量将其分为能量正平衡和能量负平衡组。两组奶牛跟踪至产后60~90d,根据有无发情表现,结合B超与直肠检查卵巢卵泡状态,最终确定了30头能量负平衡的卵巢静止奶牛为实验组(T)和30头能量正平衡的发情奶牛为健康对照组(C)。随机选取两组(T或C)奶牛各15头,应用2D-DIGE/MS技术检测两组奶牛血清样品获得两组奶牛的差异表达蛋白。通过生物信息学分析,确定差异蛋白与卵巢静止的关系。应用Western-blot技术对血清中AMBP蛋白复合体(alpha-1-microglobulin/bikunin precursor,α1微球蛋白/bikunin蛋白前体)进行验证。两组(T或C)中剩余15头奶牛,检测血清中生殖激素和AMBP蛋白复合体含量,并进行彼此的相关性分析,结合回归分析和受试者工作特征曲线(receiver operating characteristic curve,ROC),确定AMBP蛋白复合体对卵巢静止的预警作用。结果显示:(1)通过2D-DIGE/MALDI-TOF-MS检测和差异分析,发现卵巢静止组和发情组奶牛之间有12种差异表达蛋白质,其中AMBP蛋白复合体(Protein AMBP)、α-S1-酪蛋白(Alpha-S1-casein)、血红蛋白(Hemoglobin subunit beta)和结合珠蛋白(Haptoglobin)等4种蛋白表达上调,另外载脂蛋白A1(Apolipoprotein A-I)、载脂蛋白A4(Apolipoprotein A-IV)、α-1-酸性糖蛋白(Alpha-1-acid glycoprotein)、α-2-HS-糖蛋白(Alpha-2-HS-glycoprotein)、亮氨酸富集α-2-糖蛋白1(Leucine-rich alpha-2-glycoprotein 1)、补体3d(Complement component 3d(Fragment))、补体C3(Complement C3)和丝氨酸蛋白酶抑制剂E2C(Endopin 2C)等8种蛋白表达下调。通过生物信息学分析发现这些差异表达蛋白的基因功能主要与类固醇和脂质代谢,酶活性和抗氧化作用有关;参与的主要通路是脂肪和维生素消化与吸收,PPAR信号通路和补体与凝血级联。AMBP蛋白的Western-blotting结果与2D-DIGE的一致,都上调。(2)能量负平衡的卵巢静止组奶牛产后60~90d血清NEFA含量均显著高于健康发情组(p0.05),血清雌激素(Estrogen,E2)含量显著低于发情奶牛(p0.05),血清AMBP蛋白复合体浓度极显著高于健康发情组奶牛(p0.01);血清中E2浓度与试验动物分组情况呈极显著正相关(p0.01),AMBP蛋白复合体与试验动物分组呈极显著负相关(p0.01);血清NEFA与E2含量呈显著负相关(p0.01);通过回归分析和ROC分析确定了当奶牛血清中AMBP4268.59 ng/L时,奶牛患卵巢静止的风险增大。结论:本研究应用蛋白质组学技术获得了能量负平衡所致的卵巢静止奶牛血清差异表达蛋白,探讨了差异表达蛋白质与奶牛产后卵巢静止之间的关系,确定了AMBP蛋白复合体在奶牛产后卵巢静止发生的风险预警作用,为今后深入揭示能量负平衡所致的卵巢静止发生机理提供了新的方向。
[Abstract]:At present, ovarian motility has become a common postpartum follicular growth disorder in high-yielding dairy cows in intensive farms. It is characterized by a stationary state of ovaries or related ovarian structures with no periodic characteristics. However, the expression of whole body protein and its relationship with the disease have not been reported in postpartum dairy cows with ovarian motility. In this study, fluorescence Two-dimensional Differential Gel Electrophoresis (2D-DIGE) and matrix-assisted laser resolution were applied to isolate the disease. Matrix Assisted Laser Desorption/Lonization Time of Flight Mass Spectrometry (MALDI-TOF-MS) combined with bioinformatics analysis was used to obtain differentially expressed proteins in ovarian resting cow serum induced by negative energy balance. In this study, we selected the age and parity of the same type of dairy cows from an intensive dairy farm in Heilongjiang Province. According to the serum levels of beta-hydroxybutyrate (BHBA), non-esterified fatty acid (NEFA) and glucose (Glucose, Glc) from 14 to 21 days postpartum. Two groups of dairy cows were followed up to 60-90 days postpartum. According to oestrus and ovarian follicular status, 30 calorie-balanced ovarian stationary cows were selected as experimental group (T) and 30 calorie-balanced calorie cows as healthy control group (C). Fifteen dairy cows in each group (T or C) were used to detect the differentially expressed proteins in the serum of the two groups. The relationship between the differentially expressed proteins and ovarian motility was determined by bioinformatics analysis. The serum AMBP protein complex (alpha-1-microglobulin/bikunin precursor, alpha-1 microspheres) was detected by Western blot. Serum levels of reproductive hormones and AMP protein complexes were measured in 15 dairy cows in two groups (T or C), and their correlation was analyzed. Combined with regression analysis and receiver operating characteristic curve (ROC), the resting ovarian function of AMBP protein complexes was determined. The results showed that: (1) Through two-dimensional DIGE/MALDI-TOF-MS detection and differential analysis, 12 differentially expressed proteins were found between ovarian resting group and estrus group, including AMBP protein complex (Protein AMBP), alpha-S1-casein (Alpha-S1-casein), hemoglobin subunit beta (Hemoglobin subunit beta) and Haptoglobin (Haptoglobin). Apolipoprotein A-I, Apolipoprotein A-IV, Alpha-1-acid glycoprotein, Alpha-2-HS-glycoprotein, Leucine-rich alpha-2-glycoprotein 1, Complement 3D Bioinformatics analysis showed that the gene functions of these differentially expressed proteins were mainly related to steroid and lipid metabolism, enzyme activities and antioxidant activities, and the main pathways involved were fat and vitamin digestion and absorption, and PPA. Western-blotting results of AMBP protein were consistent with those of 2D-DIGE. (2) Negative energy balance ovarian motility group had significantly higher serum NEFA content than healthy estrus group (p0.05), lower serum estrogen (Estrogen, E2) content than estrus cows (p0.05), and serum AMBP protein complex. Body concentration was significantly higher than that of healthy estrous cows (p0.01); E2 concentration in serum was significantly positively correlated with the grouping of experimental animals (p0.01); AMBP protein complex was significantly negatively correlated with the grouping of experimental animals (p0.01); serum NEFA and E2 content was significantly negatively correlated (p0.01); regression analysis and ROC analysis showed that the serum of dairy cows was positively correlated with the grouping of experimental animals (p0.01). CONCLUSION: In this study, the serum differentially expressed proteins in ovarian quiescent cows induced by negative energy balance were obtained by proteomics, and the relationship between differentially expressed proteins and postpartum ovarian quiescent in dairy cows was discussed. The risk warning function of resting ovarian motility provides a new direction for further revealing the mechanism of resting ovary caused by negative energy balance.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23
本文编号：2225709
[Abstract]:At present, ovarian motility has become a common postpartum follicular growth disorder in high-yielding dairy cows in intensive farms. It is characterized by a stationary state of ovaries or related ovarian structures with no periodic characteristics. However, the expression of whole body protein and its relationship with the disease have not been reported in postpartum dairy cows with ovarian motility. In this study, fluorescence Two-dimensional Differential Gel Electrophoresis (2D-DIGE) and matrix-assisted laser resolution were applied to isolate the disease. Matrix Assisted Laser Desorption/Lonization Time of Flight Mass Spectrometry (MALDI-TOF-MS) combined with bioinformatics analysis was used to obtain differentially expressed proteins in ovarian resting cow serum induced by negative energy balance. In this study, we selected the age and parity of the same type of dairy cows from an intensive dairy farm in Heilongjiang Province. According to the serum levels of beta-hydroxybutyrate (BHBA), non-esterified fatty acid (NEFA) and glucose (Glucose, Glc) from 14 to 21 days postpartum. Two groups of dairy cows were followed up to 60-90 days postpartum. According to oestrus and ovarian follicular status, 30 calorie-balanced ovarian stationary cows were selected as experimental group (T) and 30 calorie-balanced calorie cows as healthy control group (C). Fifteen dairy cows in each group (T or C) were used to detect the differentially expressed proteins in the serum of the two groups. The relationship between the differentially expressed proteins and ovarian motility was determined by bioinformatics analysis. The serum AMBP protein complex (alpha-1-microglobulin/bikunin precursor, alpha-1 microspheres) was detected by Western blot. Serum levels of reproductive hormones and AMP protein complexes were measured in 15 dairy cows in two groups (T or C), and their correlation was analyzed. Combined with regression analysis and receiver operating characteristic curve (ROC), the resting ovarian function of AMBP protein complexes was determined. The results showed that: (1) Through two-dimensional DIGE/MALDI-TOF-MS detection and differential analysis, 12 differentially expressed proteins were found between ovarian resting group and estrus group, including AMBP protein complex (Protein AMBP), alpha-S1-casein (Alpha-S1-casein), hemoglobin subunit beta (Hemoglobin subunit beta) and Haptoglobin (Haptoglobin). Apolipoprotein A-I, Apolipoprotein A-IV, Alpha-1-acid glycoprotein, Alpha-2-HS-glycoprotein, Leucine-rich alpha-2-glycoprotein 1, Complement 3D Bioinformatics analysis showed that the gene functions of these differentially expressed proteins were mainly related to steroid and lipid metabolism, enzyme activities and antioxidant activities, and the main pathways involved were fat and vitamin digestion and absorption, and PPA. Western-blotting results of AMBP protein were consistent with those of 2D-DIGE. (2) Negative energy balance ovarian motility group had significantly higher serum NEFA content than healthy estrus group (p0.05), lower serum estrogen (Estrogen, E2) content than estrus cows (p0.05), and serum AMBP protein complex. Body concentration was significantly higher than that of healthy estrous cows (p0.01); E2 concentration in serum was significantly positively correlated with the grouping of experimental animals (p0.01); AMBP protein complex was significantly negatively correlated with the grouping of experimental animals (p0.01); serum NEFA and E2 content was significantly negatively correlated (p0.01); regression analysis and ROC analysis showed that the serum of dairy cows was positively correlated with the grouping of experimental animals (p0.01). CONCLUSION: In this study, the serum differentially expressed proteins in ovarian quiescent cows induced by negative energy balance were obtained by proteomics, and the relationship between differentially expressed proteins and postpartum ovarian quiescent in dairy cows was discussed. The risk warning function of resting ovarian motility provides a new direction for further revealing the mechanism of resting ovary caused by negative energy balance.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S858.23
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