应用蛋白质组学筛选能量负平衡卵巢静止奶牛生物标记物
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摘要
目的运用蛋白质组学技术筛选能量负平衡卵巢静止奶牛的生物标记物,建立早期诊断标准,并探讨其发病机制。方法应用荧光差异双向凝胶电泳(fluorescence two-dimensional differential gel electrophoresis,2D-DIGE)和基质辅助激光解析离子化飞行时间质谱(matrix-assisted laser desorption/lonization time of flight mass spectrometry,MALDI-TOF-MS),鉴定能量正平衡发情奶牛和能量负平衡卵巢静止奶牛的差异蛋白质。采用生物信息学、蛋白印迹和受试者工作特征(receiver operating characteristic,ROC)曲线分析差异蛋白质,并确定用于早期诊断卵巢静止的生物标志物。结果两组奶牛筛选出12种差异表达蛋白,这些差异蛋白主要参与脂类代谢、维生素代谢、补体凝血级联及过氧化物酶增殖物激活受体(peroxisome proliferator activated receptor-α,PPAR)信号通路,其中α-1-微球蛋白/bikunin前体(alpha-1-microglobulin/bikunin precursor,AMBP)可作为卵巢静止早期诊断的标志物,预警值为4 346.75 ng/L。结论应用蛋白组学筛选出能量负平衡导致奶牛卵巢静止的12种差异表达蛋白,确定了用于早期诊断卵巢静止的生物标志物,为卵巢静止的临床诊断和发病机制奠定了基础。
Objective To use proteomics techniques to screen biomarkers which negatively balance the energy in cows with inactive ovary,establish early diagnostic criteria and investigate the mechanism of pathogenesis. Methods Fluorescence two-dimensional differential gel electrophoresis(2 D-DIGE)and matrix-assisted laser desorption/ionization time-offlight mass spectrometry(MALDI-TOF-MS) were used to identify differentially expressed proteins in energy-balanced estrous cows and energy-negative balanced cows with inactive ovary. Bioinformatics,Western blot and receiver operating characteristic(ROC)curve analysis were used to analyze differential proteins and determine the biomarkers for early diagnosis of inactive ovary. Results Twelve kinds of plasma differentially expressed proteins were screened in the two groups of cows. The proteins were mainly related to lipid metabolism,vitamin digestion and absorption,complement and coagulation cascade as well as PPAR signaling pathway,among which alpha-1-microglobulin/bikunin precursor(AMBP)might be used for markers for early diagnosis of inactive ovary,with an early-warning value of 4 346. 75 ng/L. Conclusion Twelve kinds of differentially expressed proteins resulting in inactive ovary by negative energy balance in dairy cows were screened by proteomics,which was used as a biomarker for the early diagnosis of inactive ovary. It laid a foundation of clinical diagnosis and research on pathogenesis of inactive ovary.
Objective To use proteomics techniques to screen biomarkers which negatively balance the energy in cows with inactive ovary,establish early diagnostic criteria and investigate the mechanism of pathogenesis. Methods Fluorescence two-dimensional differential gel electrophoresis(2 D-DIGE)and matrix-assisted laser desorption/ionization time-offlight mass spectrometry(MALDI-TOF-MS) were used to identify differentially expressed proteins in energy-balanced estrous cows and energy-negative balanced cows with inactive ovary. Bioinformatics,Western blot and receiver operating characteristic(ROC)curve analysis were used to analyze differential proteins and determine the biomarkers for early diagnosis of inactive ovary. Results Twelve kinds of plasma differentially expressed proteins were screened in the two groups of cows. The proteins were mainly related to lipid metabolism,vitamin digestion and absorption,complement and coagulation cascade as well as PPAR signaling pathway,among which alpha-1-microglobulin/bikunin precursor(AMBP)might be used for markers for early diagnosis of inactive ovary,with an early-warning value of 4 346. 75 ng/L. Conclusion Twelve kinds of differentially expressed proteins resulting in inactive ovary by negative energy balance in dairy cows were screened by proteomics,which was used as a biomarker for the early diagnosis of inactive ovary. It laid a foundation of clinical diagnosis and research on pathogenesis of inactive ovary.
引文
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[19]郭致准.多囊卵巢综合征与环境内分泌干扰物的相关性分析及其致病机制的初步研究[D].福建厦门:厦门大学,2016.
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[2]肖鑫焕,许楚楚,李昌盛,等.能量负平衡对卵巢静止奶牛的影响[J].畜牧与饲料科学,2015,36(Z1):173-177.
[3]XU C C,XIA C,SUN Y H,et al.1H-NMR-based plasma metabolic profiling of postpartum dairy cows with ovarian inactivity[J].Acta Vet et Zoot Sin,2016,47(1):190-197.(in Chinese)许楚楚,夏成,孙雨航,等.基于核磁共振技术的卵巢静止奶牛血浆代谢谱分析[J].畜牧兽医学报,2016,47(1):190-197.
[4]NIE T T,ZHENG C Y,ZOU X,et al.Screening and identification of differential expression proteins in placenta tissue of cows with retained fetal membranes[J].Chin J Biologicals,2015,28(1):54-57.(in Chinese)聂甜甜,郑程远,邹晓,等.胎衣不下奶牛胎盘组织差异表达蛋白的筛选及鉴定[J].中国生物制品学杂志,2015,28(1):54-57.
[5]SHU S,XIA C,ZHANG H Y,et al.Biomarker discovery for cows with hypocalcemia using 2D-DIGE[J].Acta Vet et Zoot Sin,2015,46(7):1238-1245.(in Chinese)舒适,夏成,张洪友,等.基于2D-DIGE/MALDI-TOF-MS技术筛选奶牛低血钙症生物标志物[J].畜牧兽医学报,2015,46(7):1238-1245.
[6]许楚楚,李昌盛,肖鑫焕,等.黑龙江省某集约化牛场奶牛产后能量平衡与乏情的调查研究[J].畜牧与饲料科学,2015,36(z1):213-217.
[7]XU C,XIA C,SUN Y,et al.Metabolic profiles using 1 Hnuclear magnetic resonance spectroscopy in postpartum dairy cows with ovarian inactivity[J].Theriogenology,2016,86(6):1475-1481.
[8]SHU S,XIA C,ZHANG H Y,et al.Plasma proteomics analysis of dairy cows with milk fever using SELDI-TOF-MS[J].Chin J Vet Med,2016,52(4):16-18.(in Chinese)舒适,夏成,张洪友,等.基于SELDI-TOF-MS技术的奶牛乳热血浆蛋白质组学分析[J].中国兽医杂志,2016,52(4):16-18.
[9]HUANG L,YONEDA M,KIMATA K.A serum-derived hyaluronan-associated protein(SHAP)is the heavy chain of the inter alpha-trypsin inhibitor[J].J Biol Chem,1993,268(35):26725-26730.
[10]NAGYOVA E.Organization of the expanded cumulus-extracellular matrix in preovulatory follicles:a role for inter-alpha-trypsin inhibitor[J].Endocrine Regulations,2015,49(1):37-45.
[11]SUZUKI M,KOBAYASHI H,TANAKA Y,et al.Suppression of invasion and peritoneal carcinomatosis of ovarian cancer cell line by overexpression of bikunin[J].Int J Cancer,2003,104(3):289-302.
[12]WALLDIUS G,JUNGNER I.The apo B/apo A-I ratio:a strong,new risk factor for cardiovascular disease and a target for lipidlowering therapy--a review of the evidence[J].J Intern Med,2006,259(5):493-519.
[13]GOLDBERG I J,SCHERALDI C A,YACOUB L K,et al.Lipoprotein Apo C-II activation of lipoprotein lipase.Modulation by apolipoprotein A-IV[J].J Biol Chem,1990,265(8):4266-4272.
[14]王书娟,董金军,吴鹏.如何正确评价胆固醇的作用[J].实用新医学,2011.
[15]LI Y Y,LI Q F,YU W F,et al.Protective effect of vitamin C for oxidative stress on ovary exposed to dibutyl phthalate during puberty[J].J Environ Health,2017,34(1):28-32.(in Chinese)李雨颖,李群锋,余文富,等.维生素C对青春期邻苯二甲酸二丁酯暴露致大鼠卵巢氧化应激的干预作用[J].环境与健康杂志,2017,34(1):28-32.
[16]ZHAO F,DANG L Y,ZHAO X,et al.Complement and glycosylation[J].Prog in Biochem Biophysics,2017,44(10):888-897.(in Chinese)赵菲,党刘毅,赵璇,等.补体系统及其糖基化[J].生物化学与生物物理进展,2017,44(10):888-897.
[17]NILSSON E E,SAVENKOVA M I,SCHINDLER R,et al.Gene bionetwork analysis of ovarian primordial follicle development[J].PLo S One,2010,5(7):e11637.doi:10.1371/journal.pone.0011637.
[18]WANG D D,LIU Y C,ZHANG Y C,et al.Recombinant expression and functional characterization of a serpin-type serine proteinase inhibitor(Fc-serpin)from the Chinese shrimp(Fenneropenaeus chinensis)[J].J Fish China,2013,37(11):1626-1631.(in Chinese)王丹丹,刘逸尘,张亦陈,等.中国明对虾serpin型丝氨酸蛋白酶抑制剂基因的重组表达及活性分析[J].水产学报,2013,37(11):1626-1631.
[19]郭致准.多囊卵巢综合征与环境内分泌干扰物的相关性分析及其致病机制的初步研究[D].福建厦门:厦门大学,2016.
[20]HAI B,NI C M,XIE H J,et al.Association between peroxisome proliferator-activated receptor and gene-gene interactions with the apolipoprotein AⅠ/apolipoprotein B100 ratio[J].Chin J Cardiol,2015,43(4):328-333.(in Chinese)海波,倪川民,解惠坚,等.PPAR单核苷酸多态性基因-基因交互作用与载脂蛋白AⅠ/载脂蛋白B100比值的关系[J].中华心血管病杂志,2015,43(4):328-333.