临床型奶牛乳房炎乳腺组织亚细胞比较蛋白质组学研究
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摘要
本研究运用亚细胞比较蛋白质组学的研究策略和技术方法,以奶牛乳腺组织为研究模型。通过超离心技术分离出细胞核、线粒体、溶酶体、高尔基体,双向电泳分离蛋白质,PDQest7.4软件分析凝胶图谱的蛋白质表达模式,筛选差异表达蛋白斑点。质谱及生物信息学分析鉴定蛋白质,获得了一些与乳房炎发生相关的乳腺线粒体、细胞核、溶酶体、高尔基体差异表达的蛋白,并对差异表达蛋白在奶牛乳房炎发生过程中的作用机制进行了分析和探讨。
通过差速超离心将奶牛乳腺组织匀浆分离为1000g、6000g和20000g沉淀三个亚细胞组分,Nycodenz为密度梯度介质,超离心从1000g沉淀和6000g沉淀中纯化出细胞核与线粒体,20000g沉淀中纯化出溶酶体和高尔基体,酶活力检测了细胞核和线粒体的纯化倍数。结果表明:与Nycodenz纯化前相比,线粒体和细胞核的纯度提高了6倍左右。
奶牛乳腺线粒体、细胞核、溶酶体、高尔基体四个亚细胞组分的2-DE图谱分别得到583、477、509、488个左右的蛋白斑点,奶牛乳腺匀浆2-DE图谱只能得到480个左右的蛋白斑点。并发现线粒体、细胞核蛋白质在全细胞电泳图谱中基本未得到显现,亚细胞分级大大提高了低丰度蛋白质的检出效率。
奶牛乳腺组织中差异表达的蛋白是一些与细胞能量代谢、细胞迁移、分子伴侣、抗凝机制、抑制炎症反应、细胞分化、信号转导等相关蛋白,它们可能参与了机体防御、抵抗进入奶牛乳腺病原体感染的过程。研究发现乳腺处于炎症状态时代谢加快一些线粒体酶,如牛线粒体ATP酶-F1、牛心细胞色素c氧化酶、细胞色素Bc1复合体、酰基辅酶A合成酶家族3、Q-碱tRNA-核糖体转移酶、1酰胺酸磷酸核糖基转移酶等表达量上调。合成和分泌的酪蛋白(酪蛋白α- S2,酪蛋白α- s1,β-酪蛋白,β-酪蛋白A2突变体,酪蛋白-κ)表达下调,失去了稳定乳汁的能力,导致乳汁凝结。乳腺发生炎症时,乳腺代谢加快,产生的能量增多,为了避免乳腺组织细胞损伤,Hsc70的表达量急剧上升。hsp90在乳房炎发生过程中缺失,这也可能是乳房炎症发生所特有的生物标记分子,它的变化对乳房炎的反复发生可能有很大关系。膜连蛋白V、膜连蛋白A7在乳房炎发生过程中表达量上调,可能对血液凝结和炎症过程具有调节作用,可作为新的炎症调节位点。抗炎相关的白蛋白在乳房炎发生过程中表达上调,抗菌防御酶乳过氧化物酶在乳房炎乳腺表达下调;物质转运相关的蛋白质膜外孔通道蛋白,载脂蛋白A-I在乳房炎乳腺中表达下调。β-乳球蛋白,硫氧还原蛋白表达下调;锌指蛋白在乳房炎发生过程中高表达,这些差异表达的蛋白也可能是乳房炎发生潜在的生物标记分子。
通过本研究,探索了奶牛乳房炎新的诊断生物标记分子、潜在的炎症调节和治疗靶标,筛选了乳房炎抗性/易感性相关蛋白,探索其表达调控机理,为应用生物学方法预防和治疗奶牛乳房炎开辟了新途径,以及为进一步从蛋白质水平揭示、阐明奶牛乳房炎的发生机理奠定基础。
Sub-cellular comparative proteomic research strategies and techniques were applied in this study. Nucleus, mitochondria, lysosome and Golgi body were isolated from the model of mammary gland in dairy cows by ultra-centrifugation technique. Gel profiles of protein expression were analyzed and differential protein spots were screened by PDQest7.4 software. Differentially expressed proteins were analyzed and identified by Mass Spectrometry and biological informatics.Some proteins related to mastitis in mitochondria, nucleus,lysosomes and Golgi body were detected respectively, their functional mechanism were analyzed and discussed in the pathogenesis of mastitis in dairy cows.
Mammary gland tissue from Diary cows was subject to diferential centrifugation and four fractions of 1000g, 6000g and 20000g pellets were recovered. Then density gradient centrifugation was applied to the 1000g and 6000g pellets for further preparation of nuclei and mitochondrial, and 20000g pellet for preparation of lysosomes and Golgi body.The purity of nuclei and mitochondrial was increased 6-fold by this preparation.
The proteins of nuclus, mitochondrial, lysosomes and Golgi body were separated with two-dimensional gel electrophoresis (2-DE).583,477, 509and 488 protein spots were detected in the four fractions of mitochondria ,nucleus, Golgi body and lysosomes respectively, the total number of protein spots of the four fractions was about 2000, whereas just about 480 spots could be acquired for mammary gland homogenates. Since most of the mitochondrial and nuclear proteins were not able to present themselves in the 2-DE profile of the cell homogenates, more low-abundance proteins were detected through sub-cellular fractionations.
Protein changes in the mammary gland between healthy cows and clinical mastitis cow were investigated, the differentially expressed proteins were related to cell energy metabolism, cell migration, molecular chaperone, anticoagulation mechanism, inhibit inflammatory response, cell differentiation and signal transduction,which may be involved in the body defense mechanism against the mammary gland infection process. It has been found that the expression of mitochondrial enzymes such as bovine mitochondrial ATP enzyme-F1, bovine heart cytochrome c oxidase, cytochrome Bc1 complex, acyl-coenzyme A synthetase family of 3, Q-base ribosomal tRNA-transferase and 1 amide acid phosphate ribosyltransferase were up-regulated in infected mammary tissues. The expression of casein proteins (casein proteinα-S2, casein proteinα-s1,β-casein,β-casein A2 mutant protein, casein protein-κ) were down-regulated in infected mammary tissues, which may lose the ability of casein and lead to milk condensation. The expression of Hsc70 was up-regulated dramatically in mammary gland tissue infected with clinical mastitis, and may lead to speed up the energy metabolism and prevent cell from injury. Hsp90 was missed in mastitic cows, which may be related to the repeated occurrence of mastitis, and may serve as new susceptible markers of mastitis and potential protein targets for trentment. Membrane protein V and membrane protein A7 were up-regulated in mastitis cows,which may regulate blood clots and inflammatory process, and be regarded as inflammatory mediated protein and potential protein targets for mastitis treatments.Albumin-related anti-inflammatory proteins was up-regulated. Transporter protein-related substances such as external orifice of membrane channel proteins and apolipoprotein A-I were down-regulated.β-lactoglobulin and thioredoxin were down-regulated; Zinc finger protein was up-regulated,which may be potential biomarkers during mastitis in dairy cows.
In conclusion, these results may provide valuable information for the investigation of the host mammary immune system response to defense against pathogens and involving in the pathogenesis of mammary inflammation at sub-cellular level, and finding new diagnostic markers of mastitis and potential protein targets for treatment.
通过差速超离心将奶牛乳腺组织匀浆分离为1000g、6000g和20000g沉淀三个亚细胞组分,Nycodenz为密度梯度介质,超离心从1000g沉淀和6000g沉淀中纯化出细胞核与线粒体,20000g沉淀中纯化出溶酶体和高尔基体,酶活力检测了细胞核和线粒体的纯化倍数。结果表明:与Nycodenz纯化前相比,线粒体和细胞核的纯度提高了6倍左右。
奶牛乳腺线粒体、细胞核、溶酶体、高尔基体四个亚细胞组分的2-DE图谱分别得到583、477、509、488个左右的蛋白斑点,奶牛乳腺匀浆2-DE图谱只能得到480个左右的蛋白斑点。并发现线粒体、细胞核蛋白质在全细胞电泳图谱中基本未得到显现,亚细胞分级大大提高了低丰度蛋白质的检出效率。
奶牛乳腺组织中差异表达的蛋白是一些与细胞能量代谢、细胞迁移、分子伴侣、抗凝机制、抑制炎症反应、细胞分化、信号转导等相关蛋白,它们可能参与了机体防御、抵抗进入奶牛乳腺病原体感染的过程。研究发现乳腺处于炎症状态时代谢加快一些线粒体酶,如牛线粒体ATP酶-F1、牛心细胞色素c氧化酶、细胞色素Bc1复合体、酰基辅酶A合成酶家族3、Q-碱tRNA-核糖体转移酶、1酰胺酸磷酸核糖基转移酶等表达量上调。合成和分泌的酪蛋白(酪蛋白α- S2,酪蛋白α- s1,β-酪蛋白,β-酪蛋白A2突变体,酪蛋白-κ)表达下调,失去了稳定乳汁的能力,导致乳汁凝结。乳腺发生炎症时,乳腺代谢加快,产生的能量增多,为了避免乳腺组织细胞损伤,Hsc70的表达量急剧上升。hsp90在乳房炎发生过程中缺失,这也可能是乳房炎症发生所特有的生物标记分子,它的变化对乳房炎的反复发生可能有很大关系。膜连蛋白V、膜连蛋白A7在乳房炎发生过程中表达量上调,可能对血液凝结和炎症过程具有调节作用,可作为新的炎症调节位点。抗炎相关的白蛋白在乳房炎发生过程中表达上调,抗菌防御酶乳过氧化物酶在乳房炎乳腺表达下调;物质转运相关的蛋白质膜外孔通道蛋白,载脂蛋白A-I在乳房炎乳腺中表达下调。β-乳球蛋白,硫氧还原蛋白表达下调;锌指蛋白在乳房炎发生过程中高表达,这些差异表达的蛋白也可能是乳房炎发生潜在的生物标记分子。
通过本研究,探索了奶牛乳房炎新的诊断生物标记分子、潜在的炎症调节和治疗靶标,筛选了乳房炎抗性/易感性相关蛋白,探索其表达调控机理,为应用生物学方法预防和治疗奶牛乳房炎开辟了新途径,以及为进一步从蛋白质水平揭示、阐明奶牛乳房炎的发生机理奠定基础。
Sub-cellular comparative proteomic research strategies and techniques were applied in this study. Nucleus, mitochondria, lysosome and Golgi body were isolated from the model of mammary gland in dairy cows by ultra-centrifugation technique. Gel profiles of protein expression were analyzed and differential protein spots were screened by PDQest7.4 software. Differentially expressed proteins were analyzed and identified by Mass Spectrometry and biological informatics.Some proteins related to mastitis in mitochondria, nucleus,lysosomes and Golgi body were detected respectively, their functional mechanism were analyzed and discussed in the pathogenesis of mastitis in dairy cows.
Mammary gland tissue from Diary cows was subject to diferential centrifugation and four fractions of 1000g, 6000g and 20000g pellets were recovered. Then density gradient centrifugation was applied to the 1000g and 6000g pellets for further preparation of nuclei and mitochondrial, and 20000g pellet for preparation of lysosomes and Golgi body.The purity of nuclei and mitochondrial was increased 6-fold by this preparation.
The proteins of nuclus, mitochondrial, lysosomes and Golgi body were separated with two-dimensional gel electrophoresis (2-DE).583,477, 509and 488 protein spots were detected in the four fractions of mitochondria ,nucleus, Golgi body and lysosomes respectively, the total number of protein spots of the four fractions was about 2000, whereas just about 480 spots could be acquired for mammary gland homogenates. Since most of the mitochondrial and nuclear proteins were not able to present themselves in the 2-DE profile of the cell homogenates, more low-abundance proteins were detected through sub-cellular fractionations.
Protein changes in the mammary gland between healthy cows and clinical mastitis cow were investigated, the differentially expressed proteins were related to cell energy metabolism, cell migration, molecular chaperone, anticoagulation mechanism, inhibit inflammatory response, cell differentiation and signal transduction,which may be involved in the body defense mechanism against the mammary gland infection process. It has been found that the expression of mitochondrial enzymes such as bovine mitochondrial ATP enzyme-F1, bovine heart cytochrome c oxidase, cytochrome Bc1 complex, acyl-coenzyme A synthetase family of 3, Q-base ribosomal tRNA-transferase and 1 amide acid phosphate ribosyltransferase were up-regulated in infected mammary tissues. The expression of casein proteins (casein proteinα-S2, casein proteinα-s1,β-casein,β-casein A2 mutant protein, casein protein-κ) were down-regulated in infected mammary tissues, which may lose the ability of casein and lead to milk condensation. The expression of Hsc70 was up-regulated dramatically in mammary gland tissue infected with clinical mastitis, and may lead to speed up the energy metabolism and prevent cell from injury. Hsp90 was missed in mastitic cows, which may be related to the repeated occurrence of mastitis, and may serve as new susceptible markers of mastitis and potential protein targets for trentment. Membrane protein V and membrane protein A7 were up-regulated in mastitis cows,which may regulate blood clots and inflammatory process, and be regarded as inflammatory mediated protein and potential protein targets for mastitis treatments.Albumin-related anti-inflammatory proteins was up-regulated. Transporter protein-related substances such as external orifice of membrane channel proteins and apolipoprotein A-I were down-regulated.β-lactoglobulin and thioredoxin were down-regulated; Zinc finger protein was up-regulated,which may be potential biomarkers during mastitis in dairy cows.
In conclusion, these results may provide valuable information for the investigation of the host mammary immune system response to defense against pathogens and involving in the pathogenesis of mammary inflammation at sub-cellular level, and finding new diagnostic markers of mastitis and potential protein targets for treatment.
引文
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