【作者】 黄建珍；

【导师】 邹思湘；

【作者基本信息】 南京农业大学 ， 基础兽医学， 2008， 博士

【摘要】 本文以脂肪型AA肉鸡和瘦肉型三黄肉鸡为实验对象,以比较蛋白组学为主要研究手段,揭示其在胚胎发育过程中肝脏蛋白组的表达谱,并在差异比较的基础上,筛选出了影响两种肉鸡脂类代谢的关键蛋白和酶;接着利用生理调节剂——脱氢表雄酮(DHEA)在孵化前导入AA肉鸡种蛋,探索其对胚胎期肉鸡肝脏脂类代谢的影响;并结合构建肝脏蛋白质的酪氨酸磷酸化修饰谱,更加深入地揭示DHEA调控肉鸡脂类代谢的机制。1胚胎期肉鸡肝脏蛋白质表达谱的发育性变化鸡胚是脊椎动物胚体发育研究的主要模式材料,在发育生物学中具有较高的地位。肝脏在鸡胚生长发育过程中具有重要的作用,尤其是在糖代谢、脂代谢、胆汁酸的产生及造血器官的形成中发挥重大作用.本实验以生长速度快,腹部脂肪沉积较多的Arbor Acres肉鸡(简称AA肉鸡)和生长速度慢,脂肪沉积较少的三黄肉鸡(简称SH肉鸡)为实验动物,从蛋白水平探索肉鸡胚肝发育相关蛋白质的动态变化,为深入研究鸡胚肝脏的发育奠定基础。分别取9、14、19胚龄和出壳时的肝脏,经2D-PAGE分离后,对经胶体考马斯亮蓝G-250染色的图像用PDquest7.3软件进行差示分析,取差异表达的蛋白质点进行胶内酶切和MALDI-TOF-MS分析,肽指纹图谱数据检索NCBInr蛋白质数据库进行差异表达蛋白质的身份鉴定。在AA鸡和三黄鸡胚肝发育蛋白谱中,分别发现32和25个表达水平具有显著性差异变化的蛋白质点,其中26个和21个得到了鉴定。在这些被鉴定的蛋白中,大多涉及到机体内基础代谢的酶、细胞生长分化、胚胎形成及信号传导等,尤其是不同形式的硫酸基转移酶同工酶的表达呈明显的发育性变化,提示其与肉鸡胚胎的正常生长发育和肝脏功能密切相关.2两种肉鸡胚胎期肝脏脂类代谢相关蛋白表达的差异比较挑选40周龄体况良好的产蛋的脂肪型AA肉鸡和瘦肉型SH肉鸡,饲喂AA肉种鸡日粮,散养。收集种蛋孵化,分别取9、14、19胚龄和出壳时的肝脏.经2D-PAGE分离及MALDI-TOF-MS分析,确定了其中37个蛋白点的身份.结果表明,脂肪型AA肉鸡胚重、肝重均显著大于瘦肉型SH肉鸡。此外,机体内重要代谢的关键因子如磷酸烯醇式丙酮酸羧激酶、烯醇化酶、载脂蛋白A-I、脂肪酸结合蛋白、HMGCoA合成酶及硫酸基转移酶,其表达存在明显的品种差异。磷酸烯醇式丙酮酸羧激酶、载脂蛋白A-I、脂肪酸结合蛋白、HMGCoA合成酶在瘦肉型的三黄肉鸡上调表达;提示瘦肉型三黄肉鸡的糖异生作用、胆固醇代谢及脂肪酸氧化能力均强于脂肪型AA肉鸡,因而脂肪沉积减少.结果表明胚胎期肉鸡脂类代谢相关蛋白和酶的表达存在品种差异。3 DHEA对胚胎期肉鸡肝脏脂类代谢相关蛋白表达的影响DHEA是动物体内包括禽类的类固醇激素的前体.挑选蛋重相近的AA肉鸡种蛋,随机分为两组,实验组按50mg/kg蛋重导入DHEA,其溶解于50μL DMSO溶剂中,对照组则导入50μL溶剂.分别取9、14、19胚龄和刚出壳时的肝脏.经2D-PAGE分离及MALDI-TOF-MS分析,确定了其中37个蛋白点的身份。结果表明,DHEA显著降低胚胎中后期肉鸡胚重和绝对肝重。DHEA上调肝脏载脂蛋白A-I的表达,促进胆固醇转变成胆固醇酯;上调具有抗氧化作用的肝硫氧还蛋白过氧化物酶6的表达;上调肝脏硫酸基转移酶(pH5.9)的表达以促进其底物DHEA本身的生物转化;尤其重要的是,DHEA下调肝脏糖代谢中重要关键酶——丙酮酸脱氢酶的表达,减少肝细胞中合成脂肪酸的前体分子乙酰辅酶A的数量,从而有效降低肝脏脂肪的合成和转出。结果提示,DHEA可能通过对其自身的靶酶发生影响实现降脂功能.4 DHEA对胚胎期肉鸡肝脏脂类代谢相关磷酸化的研究挑选蛋重相近的AA肉鸡种蛋,随机分为两组,实验组按50mg/kg蛋重导入DHEA,其溶解于50μL DMSO溶剂中,对照组则导入50μL溶剂。分别取9、14、19胚龄和刚出壳时的肝脏.经2D-PAGE分离并结合磷酸化氨基酸抗体检测及MALDI-TOF-MS分析,确定了其中32个磷酸化蛋白点的身份,其中的22个蛋白点的磷酸化状态或磷酸化信号强度明显受DHEA的影响。结果表明,DHEA作为物质代谢调节的信号分子,通过对其自身靶酶的磷酸化/去磷酸化作用进行调节以影响其活性;DHEA增强肝脏硫氧还蛋白过氧化物酶6的磷酸化水平以提增该酶的抗氧化活性;通过提高硫酸基转移酶(pH5.9)磷酸化程度促进DHEA在肝脏中的生物转化;更重要的是揭示了DHEA减少肝脏中乙酰辅酶A的生成机制,即在下调肝脏丙酮酸脱氢酶的表达的同时增强其磷酸化程度,从而减少从糖的分解代谢向脂肪合成代谢提供乙酰辅酶A原料,进而减少脂肪的合成。更多还原

【Abstract】 In order to research the changes of proteins profiles and the differences of lipids metabolism in the embryonic broiler chickens liver,a comparative proteomics strategy was applied and AA broiler and Sanhuang broiler with different growth and fat deposition were employed in present study.At the same time,in the comparison of expressions of proteins related to lipids metabolism in two broiler chickens liver,we had sieved the key proteins and enzymes which resulted in the differences of lipids metabolism in two broiler chickens liver.Subsequently,as a physiological regulator,DHEA was injected to AA fertilized eggs before incubation to explore the lipids metabolism-regulating mechanism of it.In order to identify phosphoproteins and investigate their alteration in phosphorylation status associated with DHEA function,AA chicken embryo’ liver phosphoproteome was analyzed using 2-DE in combination with western blotting of specific anti-phosphotyrosine.This result of this study provided a new rode for the lipids metabolism-regulating mechanism in broiler chickens.1 Developmental Change of Proteins Profiles in the Embryonic Broiler Chickens LiverThe chicken embryo has been used widely as an experimental model for studying development.The embryonic liver plays a crucial role in metabolism and provides many essential functions.The most common and best-characterized functions include bile production,lipid metabolism,hematopoietic organogenesis and glucose metabolism.In order to research the development pattern,AA broiler and SH broiler were employed in present study.AA chickens are the fat meat-type ones,which have been used to perform the genetic analysis and investigate the development of embryonic chicken liver.SH broiler chicken is a famous Chinese indigenous breed,with uniform fat in body,high intramuscle fat and delicious meat.Proteomic analysis was used to study the molecular mechanism of chicken embryonic liver development.We collected embryonic livers from AA and SH chicken embryos on 9,14,19 days of incubation and hatching.Proteins were extracted and fractionated by 2-DE.Neuhoff’s colloidal Coomassie Blue G-250 stain was carried out. Stained gels were scanned and analyzed using PDQuest 7.3 software(Bio-Rad).In-gel trypsin digestion of the differential protein spots,MALDI-TOF-MS was analyzed. Twenty-one and Twenty-six proteins of AA and SH chicken embryonic liver were identified to participate in such processes,including carbohydrate metabolism,cell division, lipid metabolism and signal transduction,especially,two kinds of isozymes of sulfotransferase were detected and their expression patterns exhibited obvious changes among the developmental stages of the liver of broiler chicken embryo.This suggested that sulfotransferase had an important role in the liver function,growth and development of broiler chicken embryo.Our results provide insight into biochemical events taking place during the development of AA and SH chicken embryonic liver.2 Comparison of Expressions of Proteins Related to Lipids Metabolism in Two Broiler Chickens Liver with Different Fat Deposition at Embryonic StageIn order to investigate the difference of fat metabolism during embryonic development, AA broiler and Sanhuang broiler chickens with different growth and fat deposition were employed in present study.They were fed with the same diet and fioor-reared.The liver were gained on days 9,14 and 19 of embryonic development as well as at hatching. Proteins were extracted and fractionated by 2-DE and identified by MALDI-TOF-MS. Peptide mass fingerprinting of the differentially-expressed proteins was performed using the server of MASCOT or Prosperctor or ProFound.Thirty-seven proteins were identified. This study showed that embryonic weight,liver weight increased with development proceeding.Of the identified 37 differential proteins,phosphoenolpyruvate carboxykinase, apolipoprotein A-I,Fatty acid-binding protein(L-FABP), 3-hydroxy-3-methylglutaryl-Coenzyme A synthase were up-regulated in SH chickens.This suggested that the gluconeogenesis,cholesterol metabolism and fatty acids oxidation in the livers of SH chickens were higher than that in the livers of AA chickens.So the differential expression of the L-FABP could have an effect on fat deposition in the chicken.This suggested that some proteins and enzymes of lipids metabolism existed breed character.3 Effect of DHEA on the Expressions of the Proteins Related to Lipids Metabolism in Broiler Chickens Embryo’ Liver during Embryonic DevelopmentDehydroepiandrosterone is a precursor of steroid secreted by the adrenal cortex in animal including poultry.In order to explore the possible fat-reducing mechanism of DHEA,this study was to investigate the effect of in ovo administration of DHEA on lipids metabolism and the regulating mechanism of DHEA on lipids metabolism in broiler chickens during embryonic development.The treatment was injected 50μL DMSO cotained 50 mg DHEA per kilogram eggs and the control was only injected 50μL DMSO to fertilized eggs.The livers were gained on days 9,14 and 19 of embryonic development as well as at hatching.The extracted proteins were analyzed using 2-DE.The differential spots were identified by MALDI-TOF-MS.Peptide mass fingerprinting of the differentially-expressed proteins was performed using the server of MASCOT or Prosperctor or ProFound.Thirty-seven proteins were identified.This result showed that embryonic weight and liver weight in DHEA group was significantly lower than that in control group.The expression of apolipoproteinA-I was increased in DHEA group,which facilitated cholesterol to cholesterol esters;the expression of peroxiredoxin 6 which had antioxidative activity was increased in DHEA group;at the same time,sulfotransferase (according to pH5.9) was increased in DHEA group,this demonstrated that DHEA was the best substrate for sulfotransferase(according to pH5.9) in broilers.Above all,the expression of pyruvate dehydrogenase was decreased in DHEA group.This suggested that DHEA reduced the supply of acyl coenzyme A by decreasing the activity of pyruvate dehydrogenase so as to decline the deposition of fat in broiler chicken during embryonic development.4 Study of DHEA on the Expressions of Tyrosine Phosphorylation Proteins Related to Lipids Metabolism in Broiler Chickens Embryo’ Liver In order to explore the possible fat-reducing mechanism of DHEA,this study was to investigate the effect of DHEA on lipids metabolism and the regulating mechanism of DHEA on lipids metabolism in broiler chickens during embryonic development.The treatment was injected 50μL DMSO cotained 50 mg DHEA per kilogram eggs and the control was only injected 50μL DMSO to fertilized eggs.The livers were gained on days 9, 14 and 19 of embryonic development as well as at hatching.The extracted proteins were analyzed using 2-DE in combination with Western blotting of specific anti-phosphotyrosine. The differential spots were identified by MALDI-TOF-MS or MALDI-TOF-TOF-MS. Peptide mass fingerprinting of the differentially-expressed proteins was performed using the server of MASCOT or Prosperctor or ProFound.Thirty-two tyrosine phosphorylation proteins were identified.Of which,22 tyrosine phosphorylation proteins showed significantly difference in volume between the control and the treatment.This result showed that DHEA,as the signaling molecule of substance metabolism,had regulated the activity of its target enzyme by the phosphorylation/dephosphorylation;DHEA increased the tyrosine phosphorylation proteins of peroxiredoxin 6 so as to enhanced its antioxidative activity;DHEA increased the tyrosine phosphorylation proteins of sulfotransferase (according to pH5.9) so as to promote the biotransformation of DHEA;what’s more, DHEA reduced the supply of acyl coenzyme A by decreasing the activity of pyruvate dehydrogenase so as to decline the deposition of fat in broiler chicken during embryonic development.This could be one of the fat-reducing mechanisms of DHEA.更多还原