正常及早期糖尿病大鼠视网膜的蛋白组学初步研究
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摘要
糖尿病及其并发症的防治是关乎国计民生的重大课题,糖尿病眼部并发症占到糖尿病并发症的20%～24%,其中糖尿病视网膜病变(Diabetic Retinopathy,DR)是最主要的致盲原因。长久以来DR的血管病变机制一直是临床防治及基础研究的重点。然而近十年来DR的发病机制研究已逐渐转向了对早期糖尿病视网膜神经退行性变原因的探讨。糖尿病作为一种多因素的全身性疾病,高糖代谢、超氧化物和糖基化终末产物(AGEs)等代谢毒性物质的积聚、缺血缺氧、氧化应激、炎症及免疫反应等等均可最终导致神经组织凋亡、胶质增生等病理改变。糖尿病发病伊始有哪些蛋白因子作为“扳机”触发了这些病理改变呢?随着病程的延长,又有哪些因子参与进来呢?它们之间有哪些联系呢?近年来迅速发展的蛋白质组学研究,具有观察由多基因事件引起的多蛋白质组分整体变化的独特优势,更接近生命现象的本质,能动态、整体、定量地考察疾病发生发展过程中全部蛋白质种类和数量的变化,对于探讨疾病机理、寻找疾病诊断的特异标志物和药物治疗的靶标来说,是一种有效的高通量的研究模式,可获得一些传统手段无法得到的新蛋白标志物和关键分子,极大地丰富诊断标志物的选择组合,并有助于复杂致病机理的全面阐明,目前已被广泛用于研究各种疾病的发生、发展规律,并取得了很多重大的成就。
本硕士研究生论文工作是复旦大学眼耳鼻喉科医院玻璃体视网膜疾病课题组与复旦大学上海医学院生物医学平台的合作项目。本研究工作的主要贡献为:采用STZ诱导的糖尿病大鼠模型,对成模早期(成模后1,2,4,6,8周内)5组时点及正常大鼠的神经视网膜进行了蛋白组学液相色谱串联质谱分析。研究初步建立了基本的正常大鼠视网膜的蛋白谱,以此作为研究早期糖尿病大鼠视网膜蛋白表达差异的基础线,我们对各时点比较后得到差异蛋白,筛选早期糖尿病视网膜神经病变相关因子,同时按早期糖尿病病程建立时间梯度曲线,从而为进一步纵向功能性研究打下基础。为临床糖尿病视网膜病变的早期防治提供了新思路。
本论文由四部分组成。第一章前言首先概述了早期糖尿病视网膜病变机制的研究进展,提出早期糖尿病视网膜即可出现神经退行性变,其病变机制是由多因素、多通路控制的,故而需要引入蛋白组学为研究开拓思路,提供新线索。然后概述了蛋白质组学这一新兴学科发展现状和技术进展,包括双相凝胶电泳技术,生物质谱技术,色谱分离技术,蛋白质芯片技术等。然后概述了蛋白组学在糖尿病视网膜病变机制研究中的应用进展,提出本课题工作的研究方向。
第二章所述工作为论证蛋白组学研究视网膜样本的可行性,以及选择恰当的蛋白组学研究方案,先用正常大鼠神经视网膜样品进行蛋白组学预试验。本实验中共从6只SD大鼠、12只眼的神经视网膜中提取到了蛋白3 mg,进行2-PADE电泳跑胶,考马斯亮蓝染色后得到了清晰的条带和蛋白点。故提示大鼠视网膜的蛋白组研究对蛋白样品量要求至少达到3mg以上,才能得到较清晰的结果。也就是实验设计中要求每组样品至少有6只以上大鼠的双眼视网膜方可达到蛋白组分析的定量要求。为了提高研究的敏感性及特异性,最终决定采用蛋白组学液相色谱串联质谱分析法(SCX-RPLC-MSMS)作为研究方法。
第三章所述工作用高剂量(65mg/kg)STZ成功建立了Ⅰ型糖尿病大鼠模型,共有43只大鼠成模,造模成功率为74.1%。大鼠模型表现出典型Ⅰ型糖尿病症状。5组糖尿病大鼠自成模后血糖浓度保持在16.7mmol/L以上,并且较稳定。对照组血糖浓度无明显波动,基本在5.88±0.01mmol/L左右。糖尿病大鼠体重随着日龄的增加而减轻,对照组的体重随着日龄的增加而增加。本实验所用的模型为糖尿病模型造模成功后8周、6周、4周、2周及1周内的大鼠,为目前公认的早期糖尿病大鼠模型。
第四章所述工作建立了正常大鼠神经视网膜全蛋白质表达谱并且以正常组为基线,对5组时点的早期糖尿病大鼠神经视网膜进行了液相色谱串联质谱分析。共获得207个差异蛋白的定性定量信息。这些差异蛋白中有11个仅存在于正常大鼠视网膜中,其余为正常及早期糖尿病大鼠视网膜所重叠共有,并且随病程的发展其表达在各时点有所不同。其中又有177个从发病后一周及两周后就出现了显著的表达变化,并且随病程的发展呈现不同的变化曲线。这些差异蛋白涉及多种蛋白功能,如细胞结构、信号转导、代谢调控、应激反应、神经元功能、视觉功能等等。由于蛋白数量庞大,蛋白的分类及功能论证工作尚未完成,故而无法对所有差异蛋白进行全面阐述。本文仅着重探讨了以下几种具有重要意义的蛋白:高移动族核小体结合域家族的Hmgn1、酪氨酸3单加氧酶(Ywhag 14-3-3 protein)、神经元蛋白激酶酪氨酸底物(Pacsin1,Protein kinase C and caseinkinase substrate in neurons protein 1)以及水通道蛋白—4(AQP4 IsoformShort of Aquaporin-4)。它们随病程发展有不同的表达变化,并且与早期糖尿病的视网膜病变机制有着密切关系。本工作建立了正常SD大鼠神经视网膜的蛋白质组数据库,其中包含386个非冗余的蛋白质,这一数据库对大鼠视网膜相关研究具有较高的参考价值。
The controlling of diabetes and its complications is an important issue for the public health and the development of all the countries around the world.About 20%to 24%of these complications affect eyes.Furthermore, the diabetic retinopathy(DR) is the major cause of blindness nowadays.A lot of papers has been reported which focused on the mechanism of vascular pathological changes of DR.However,the hotspot of the discussions and controversies have turned to the neuropathy happens in the early-staged diabetic retina.Diabetes is a multi-factored and a systemic disease concerning a mass of pathological courses such as the accumulation of the toxic products of metabolism disorder,ischemia, anoxia,oxidative stress responses,inflammatory and immune disorders. All of the courses above will finally cause neural apoptosis and the glial hyperplasia which character the retinal neuropathy.So here comes the question:which protein is the trigger of this pathological cascade? Is there any other protein makes a chain of the cascade? What' s the relation-ship among those factors?
Recent technological advancements in proteomics allow us to study global patterns of protein content and activity and how these change during development or in response to disease,it has boosted our understanding of systems-level cellular behavior and mechanism of disease. In addition,proteomics benefits the identification of new drug targets and the development of new diagnostic markers in clinical research.
The main outcome of this paper is the basic proteome map of normal rat retina and comparison with the proteome of early-staged(stage divided into lweek,2 weeks,4 weeks,6 weeks and 8 weeks after the onset of the diabetes) STZ-induced diabetic rat retina by using the SCX-RPLC-MSMS techniques.Our study provided the global information of proteomic alteration during the pathological course of the early-staged diabetes, the proteome map of normal rat retina as the baseline.These results uncover a lot of factors which are related to the retinal neuropathy and vascular changes,as well.Moreover,we could draw some curves of these alterations according to the progression of diabetes.The study provides us a new understanding of the pathogenesis of DR.
This paper is composed with four parts.PartⅠis an overview of the papers of the pathogenesis of early-staged DR and the development of the proteomics techniques concerning two dimensional polyacrylamide gel electrophoresis(2D-PAGE),liquid chromatography(LC),mass spectrometry and microarray,etc.And then we introduce the applications of proteomics in the pathogenesis studies of DR,the purpose of our study following.
PartⅡis the preparation work before the launch of the study.We gained 3mg of protein from the retinas of 6 normal SD rats.Protein were separated with 2-DE.The gels were stained by comassia brilliant blue and silver stain.The protein spots were clear.It is reported that proteomics is highly limited by the dose of protein and the purity of the sample. The result proved that the proteomics study of the rat retina is feasible although retinal is a kind of tiny and complicated tissue.We finally chose SCX-RPLC-MSMS as the major study method in order to improve the sensitivity and accuracy of the experiment.
PartⅢis the establishment of the type I diabetic animal models. 43 SD rats were considered to be diabetic rats after the injection of STZ and took on the typical symptoms of type I DM.And the glucose dose of the placebo group was normal which was among 5.88±0.01mmol/L.The diabetic rats were losing weight along with their aging.The animal models were early-staged diabetic rats which were divided into 5 groups by the course of diabetes(1 week,2 weeks,4 weeks,6 weeks and 8 weeks).
The last part work provides us a global proteome profile of normal rat retina containing 386 spots of protein and a differential expression profile of the early-staged diabetic rat retina.The differential expression profile contains 207 spots of protein,11 among which just exist in the normal rat retina.And 177 of them had some apparent changes of expression just after the onset of diabetes,early as 1 week to 2 weeks. We could draw curves of these changes according to time-point of the disease.Work has not been totally accomplished since the huge profile contains a mass of protein with different functions,such as house keeping, structure,cell signaling,metabolism,stress response,visual and neuronal functions.Hence,further study is needed.In this paper we mainly discussed some protein with close relations with the pathogenesis of early-staged DR,such as Hmgnl,Ywhag 14-3-3 protein,Pacsinl,and AQP4, etc.Furthermore,the global proteome profile of the normal rat retina will provide a reference to the future study of retinal diseases.
本硕士研究生论文工作是复旦大学眼耳鼻喉科医院玻璃体视网膜疾病课题组与复旦大学上海医学院生物医学平台的合作项目。本研究工作的主要贡献为:采用STZ诱导的糖尿病大鼠模型,对成模早期(成模后1,2,4,6,8周内)5组时点及正常大鼠的神经视网膜进行了蛋白组学液相色谱串联质谱分析。研究初步建立了基本的正常大鼠视网膜的蛋白谱,以此作为研究早期糖尿病大鼠视网膜蛋白表达差异的基础线,我们对各时点比较后得到差异蛋白,筛选早期糖尿病视网膜神经病变相关因子,同时按早期糖尿病病程建立时间梯度曲线,从而为进一步纵向功能性研究打下基础。为临床糖尿病视网膜病变的早期防治提供了新思路。
本论文由四部分组成。第一章前言首先概述了早期糖尿病视网膜病变机制的研究进展,提出早期糖尿病视网膜即可出现神经退行性变,其病变机制是由多因素、多通路控制的,故而需要引入蛋白组学为研究开拓思路,提供新线索。然后概述了蛋白质组学这一新兴学科发展现状和技术进展,包括双相凝胶电泳技术,生物质谱技术,色谱分离技术,蛋白质芯片技术等。然后概述了蛋白组学在糖尿病视网膜病变机制研究中的应用进展,提出本课题工作的研究方向。
第二章所述工作为论证蛋白组学研究视网膜样本的可行性,以及选择恰当的蛋白组学研究方案,先用正常大鼠神经视网膜样品进行蛋白组学预试验。本实验中共从6只SD大鼠、12只眼的神经视网膜中提取到了蛋白3 mg,进行2-PADE电泳跑胶,考马斯亮蓝染色后得到了清晰的条带和蛋白点。故提示大鼠视网膜的蛋白组研究对蛋白样品量要求至少达到3mg以上,才能得到较清晰的结果。也就是实验设计中要求每组样品至少有6只以上大鼠的双眼视网膜方可达到蛋白组分析的定量要求。为了提高研究的敏感性及特异性,最终决定采用蛋白组学液相色谱串联质谱分析法(SCX-RPLC-MSMS)作为研究方法。
第三章所述工作用高剂量(65mg/kg)STZ成功建立了Ⅰ型糖尿病大鼠模型,共有43只大鼠成模,造模成功率为74.1%。大鼠模型表现出典型Ⅰ型糖尿病症状。5组糖尿病大鼠自成模后血糖浓度保持在16.7mmol/L以上,并且较稳定。对照组血糖浓度无明显波动,基本在5.88±0.01mmol/L左右。糖尿病大鼠体重随着日龄的增加而减轻,对照组的体重随着日龄的增加而增加。本实验所用的模型为糖尿病模型造模成功后8周、6周、4周、2周及1周内的大鼠,为目前公认的早期糖尿病大鼠模型。
第四章所述工作建立了正常大鼠神经视网膜全蛋白质表达谱并且以正常组为基线,对5组时点的早期糖尿病大鼠神经视网膜进行了液相色谱串联质谱分析。共获得207个差异蛋白的定性定量信息。这些差异蛋白中有11个仅存在于正常大鼠视网膜中,其余为正常及早期糖尿病大鼠视网膜所重叠共有,并且随病程的发展其表达在各时点有所不同。其中又有177个从发病后一周及两周后就出现了显著的表达变化,并且随病程的发展呈现不同的变化曲线。这些差异蛋白涉及多种蛋白功能,如细胞结构、信号转导、代谢调控、应激反应、神经元功能、视觉功能等等。由于蛋白数量庞大,蛋白的分类及功能论证工作尚未完成,故而无法对所有差异蛋白进行全面阐述。本文仅着重探讨了以下几种具有重要意义的蛋白:高移动族核小体结合域家族的Hmgn1、酪氨酸3单加氧酶(Ywhag 14-3-3 protein)、神经元蛋白激酶酪氨酸底物(Pacsin1,Protein kinase C and caseinkinase substrate in neurons protein 1)以及水通道蛋白—4(AQP4 IsoformShort of Aquaporin-4)。它们随病程发展有不同的表达变化,并且与早期糖尿病的视网膜病变机制有着密切关系。本工作建立了正常SD大鼠神经视网膜的蛋白质组数据库,其中包含386个非冗余的蛋白质,这一数据库对大鼠视网膜相关研究具有较高的参考价值。
The controlling of diabetes and its complications is an important issue for the public health and the development of all the countries around the world.About 20%to 24%of these complications affect eyes.Furthermore, the diabetic retinopathy(DR) is the major cause of blindness nowadays.A lot of papers has been reported which focused on the mechanism of vascular pathological changes of DR.However,the hotspot of the discussions and controversies have turned to the neuropathy happens in the early-staged diabetic retina.Diabetes is a multi-factored and a systemic disease concerning a mass of pathological courses such as the accumulation of the toxic products of metabolism disorder,ischemia, anoxia,oxidative stress responses,inflammatory and immune disorders. All of the courses above will finally cause neural apoptosis and the glial hyperplasia which character the retinal neuropathy.So here comes the question:which protein is the trigger of this pathological cascade? Is there any other protein makes a chain of the cascade? What' s the relation-ship among those factors?
Recent technological advancements in proteomics allow us to study global patterns of protein content and activity and how these change during development or in response to disease,it has boosted our understanding of systems-level cellular behavior and mechanism of disease. In addition,proteomics benefits the identification of new drug targets and the development of new diagnostic markers in clinical research.
The main outcome of this paper is the basic proteome map of normal rat retina and comparison with the proteome of early-staged(stage divided into lweek,2 weeks,4 weeks,6 weeks and 8 weeks after the onset of the diabetes) STZ-induced diabetic rat retina by using the SCX-RPLC-MSMS techniques.Our study provided the global information of proteomic alteration during the pathological course of the early-staged diabetes, the proteome map of normal rat retina as the baseline.These results uncover a lot of factors which are related to the retinal neuropathy and vascular changes,as well.Moreover,we could draw some curves of these alterations according to the progression of diabetes.The study provides us a new understanding of the pathogenesis of DR.
This paper is composed with four parts.PartⅠis an overview of the papers of the pathogenesis of early-staged DR and the development of the proteomics techniques concerning two dimensional polyacrylamide gel electrophoresis(2D-PAGE),liquid chromatography(LC),mass spectrometry and microarray,etc.And then we introduce the applications of proteomics in the pathogenesis studies of DR,the purpose of our study following.
PartⅡis the preparation work before the launch of the study.We gained 3mg of protein from the retinas of 6 normal SD rats.Protein were separated with 2-DE.The gels were stained by comassia brilliant blue and silver stain.The protein spots were clear.It is reported that proteomics is highly limited by the dose of protein and the purity of the sample. The result proved that the proteomics study of the rat retina is feasible although retinal is a kind of tiny and complicated tissue.We finally chose SCX-RPLC-MSMS as the major study method in order to improve the sensitivity and accuracy of the experiment.
PartⅢis the establishment of the type I diabetic animal models. 43 SD rats were considered to be diabetic rats after the injection of STZ and took on the typical symptoms of type I DM.And the glucose dose of the placebo group was normal which was among 5.88±0.01mmol/L.The diabetic rats were losing weight along with their aging.The animal models were early-staged diabetic rats which were divided into 5 groups by the course of diabetes(1 week,2 weeks,4 weeks,6 weeks and 8 weeks).
The last part work provides us a global proteome profile of normal rat retina containing 386 spots of protein and a differential expression profile of the early-staged diabetic rat retina.The differential expression profile contains 207 spots of protein,11 among which just exist in the normal rat retina.And 177 of them had some apparent changes of expression just after the onset of diabetes,early as 1 week to 2 weeks. We could draw curves of these changes according to time-point of the disease.Work has not been totally accomplished since the huge profile contains a mass of protein with different functions,such as house keeping, structure,cell signaling,metabolism,stress response,visual and neuronal functions.Hence,further study is needed.In this paper we mainly discussed some protein with close relations with the pathogenesis of early-staged DR,such as Hmgnl,Ywhag 14-3-3 protein,Pacsinl,and AQP4, etc.Furthermore,the global proteome profile of the normal rat retina will provide a reference to the future study of retinal diseases.
引文
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19. N. Adachi-Uehara et al. Up-regulation of genes for oxidative phosphorylation and protein turnover in diabetic mouse retina. Experimental Eye Research. 2006, 83:849-857.
20. Miyamoto K, Khosrof S, Bursell S-E, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc Natl Acad Sci USA. 1999, 96:10836-10841.
21. Zhang J, Gerhardinger C, Lorenzi M. Early complement activation and decreased levels of glycosylphosphatidylinositol-anchored complement inhibitors in human and experimental diabetic retinopathy.Diabetes. 2002, 51:3499-3504.
22. Rungger-Bra¨ndle E, Dosso AA, Leuenberger PM. Glial reactivity an early feature of diabetic retinopathy. Invest Ophthalmol Vis Sci. 2000, 41:1971-1980.
23. Carmo A, Cunha-Vaz JG, Carvalho AP, Lopes MC. L-arginine transport in retinas from strep-tozotocin diabetic rats: correlation with the level of IL-1_ and NO synthase activity. Vision Res. 1999, 39:3817-3823.
24. Gerhardinger C, Costa MB, Coulombe MC. Acute-Phase Response Proteins in Diabetic Retinopathy. IOVS. 2005, 46 (1)
25. King GL, Brownlee M, et al. The cellular and molecular mechanisms of diabetic complications. Endocrinol. Metab. Clin. N. Am. 1996, 25 :255-270.
26. Fields S. Proteomics in Genome land. Science. 2001,16(291): 1221-1224 [DOI: 10.1126/science.291.5507.1221]
27. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis. 1995,16:1090-1094
28. Anderson NG, Anderson NL, Human Proteins Index Project. Behring Inst. Mitt. 1979, 63: 169-210.
29. Kenyon G, et al. Defining the mandate of proteomics in the post-genomics era: workshop report.Mol Cell Proteomics. 2002,1:765-780.
30. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975, 250:4007-4021.
31. Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik. 1975, 26:231-243.
32. Bjellqvist B, Ek K, Righetti PG, et al. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys Methods. 1982, 6: 317-339.
33. Grog A, Obermaier C, Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 2000, 21:1037-1053.
34. Corthals GL, et al. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis. 2000, 21:1104-1115.
35. Washburn MP, Wolters D, Yates JR III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001,19:242-247.
36. Fenn JB, et al. Electrospray ionization for large biomolecules. Science.1989, 246:64-71.
37. Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988, 60:2299-2301.
38. Shen Y, et al. Nanoscale proteomics. Anal Bioanal Chem. 2004, 378:1037-1045.
39. Link AJ, et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999, 17:676-682.
40. Bresgen M, Baum U,Esser P, et al. Protein composition of the vitreous body in prolifertative diabetic retinopathy. An analysis with 2-D-electrophoresis. Ophthalmology. 1994, 91: 758-762.
41. Ouchi M, West K, Crabb JW, et al. Proteomic analysis of vitreous from diabetic macular edema. Experimental Eye Research. 2005, 81:176-182.
42. Funatsu H, Yamashita H, Ikeda T, et al. Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with diabetic macular edema and other retinal disorders. Am. J. Ophthalmol. 2002,133: 537-543.
43. Gao BB, Clermont A, Rook S, et al. Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nat Med. 2007,13(2): 181-8.
44. Garcia RM, Canals F, Hernandez C, et al. Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of Proliferative diabetic retinopathy. Diabetologia. 2007 Jun, 50(6):1294-303.
45. Quin GJ, Len AC, Billson FA, et al. Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: New insight in the pathogenesis of diabetic retinopathy. Proteomics. 2007, 7: 2636-2650.
46. Li Q, Puro DG, et al. Diabetes-induced dysfunction of the glutamate transporter in retinal Muller cells. IOVS. 2002, 43(9):3109-3116.
47. Lieth E, LaNoun KF, Antonetti DA, et al. Diabetes reduces glutamate oxidation and glutamine synthesis in the retina. Exp Eye Res. 2000, 70:723-730.
48. Liu Y, Huang MH, et al. Screening of the genes related to CNS development and injury by high density genechips. ACTA. 2004, 26(24)
49. Takagi C, Bursell SE, Lin YW, et al. Regulation of retinal hemodynamics in diabetic rats by increased expression and action of endothelin-1. IOVS.1996, 37:2504-2518.
50. Ishii H, Jirousek MR, Koya D, et al. Amelioration of vascular dysfunctions in diabetic rats by an oral PKCβ inhibitor. Science. 1996, 272:728-731.
51. Kim YH, Kim YS, et al. Expression of 14-3-3 zeta and interaction with protein kinase C in the rat retina in early diabetes. Diabetologia. 2005, 48(7):1411-1415.
52. Boyd-Kimball D, Castegna A, Sultana R, et al. Proteomic identification of proteins oxidized by Abeta (1-42) in synaptosomes: implications for Alzheimer's disease. Brain Res. 2005, 24, 1044(2):206-215.
53. LinksSultana R, Boyd-Kimball D, Poon HF, et al. Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD.Neurobiol Aging. 2006, 27 (11):1564-1576
54. Hong LE, Wonodi I, Avila M, et al. Dihydropyrimidinase-related protein 2 (DRP-2) gene and association to deficit and nondeficit schizophrenia. Am J Med Genet B Neuropsychiatr Genet. 2005,136(1):8-11.
55. Greene ND, Bamidele A, Choy M, et al. Proteome changes associated with hippocampal MRI abnormalities in the lithium pilocarpine-induced model of convulsive status epilepticus. Proteomics. 2007, 7(8): 1336-1344.
56. Perluigi M, Fai Poon H, Hensley K, et al. Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice-a model of familial amyo-trophic lateral sclerosis. Free Radic Biol Med. 2005,1, 38(7): 960-968.
57. Chen A, Liao WP, Lu Q, et al. Upregulation of dihydropyrimidinase-related protein 2, spec-trin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats--a proteomics approach. Neurochem Int. 2007, 50(7-8): 1078-1086.
58. Nagelhus EA, Veruki ML, Torp R, et al. Aquaporin-4 water channel protein in the rat retina and optic nerve: polarized expression in Muller cells and fibrous astrocytes. J Neurosci. 1998,18: 2506-2519.
59. Hamann S, Zeuthen T, La Cour M, et al. Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye. Am J Physiol. 1998, 274:1332-1345.
60. Manley GT, Fujimura M, Ma T, et al. Aquaporin-4 deletion in mice reduces brain edema following acute water intoxication and ischemic stroke. Nat Med. 2000, 6:159-163.
61. Newman EA. Regulation of extracellular and pH by polarized ion fluxes in glial cells: the retinal Muller cell. Neuroscientist. 1996, 2:1174-1175.
62. Nagelhus EA, Horio Y, Inanobe A, et al. Immunogold evidence suggests that coupling of K siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains. Glia. 1999, 26:47-54.
63. Antcliff RJ, Marshall J, et al. The pathogenesis of edema in diabetic maculopathy. Semin Ophthalmol. 1999,14: 223-232.
64. Quagliarello VJ, Wispelwey B, Long Jr WJ, et al. Recombinant human interleukin-1 induces meningitis and blood-brain barrier injury in the rat: characterization and comparison with tumor necrosis factor. Journal of Clinical Investigation. 1991, 87(4): 1360-1366.
65. Kowluru RA, Odenbach S. Role of interleukin-1β in the pathogenesis of diabetic retinopathy. British Journal of Ophthalmology, 2004, 88(10): 1343-1347.
66. Du Y, Sarthy VP, Kern TS. Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats. American Journal of Physiology. 2004, 287: 735-741.
67. Vincent JA, Mohr S. Inhibition of caspasel/interleukin-1β signaling prevents degeneration of retinal capillaries in diabetes and galactosemia. Diabetes. 2007, 56(1): 224-230.
68 Ayalasomayajula SP, Amrite AC, Kompella UB, et al. Inhibition of cyclooxygenase-2, but not cyclooxygenase-1, reduces prostaglandin E2 secretion from diabetic rat retinas. European Journal of Pharmacology. 2004, 498(1-3): 275-278.
69. Ayalasomayajula SP, Kompellal UB. Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozoto-cin-induced diabetic rat model. European Journal of Pharmacology. 2003,458(3): 283-289.
70. Joussen AM, Poulaki V, Mitsiades N, et al. Nonsteroidal anti-inflammatory drugs prevent early diabetic retinopathy via TNF-α suppression. The FASEB Journal. 2002,16(3): 438-440.
71. Kern TS, Miller CM, Du Y, et al. Topical administration of nepafenac inhibits diabetes induced retinal microvascular disease and underlying abnormalities of retinal metabolism and physiology. Diabetes. 2007, 56(2): 373-379.
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[1]Fields S.Proteomics in Genome land.Science 16 February 2001 291:1221-1224[DOI:10.1126/science.291.5507.1221]
[2]Wasinger VC,Cordwell SJ,Cerpa-Poljak A,et al.Progress with gene-product mapping of the Mollicutes:Mycoplasma genitalium.Electrophoresis,1995,16:1090-1094
[3]Anderson NG,Anderson NL,Human Proteins Index Project.Behring Inst.Mitt.1979;63:169 -210.
[4]Kenyon G,et al.Defining the mandate of proteomics in the post-genomics era:workshop report.Mol Cell Proteomics.2002;1:765 -780.
[5]O' Farrell PH.High resolution two-dimensional electrophoresis of proteins.J Biol Chem.1975;250:4007 - 4021.
[6]Klose J.Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues.A novel approach to testing for induced point mutations in mammals.Humangenetik.1975;26:231 -243.
[7]Bjellqvist B,Ek K,Righetti PG,et al.Isoelectric focusing in immobilized pH gradients:principle,methodology and some applications.J Biochem Biophys Methods,1982,6:317-339.
[8] Grog A, Obermaier C, Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis, 2000, 21: 1037-1053.
[9] Corthals GL, et al. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis. 2000; 21: 1104 — 1115.
[10] Washburn MP, Wolters D, Yates JR III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001:19:242-247.
[11] Fenn JB, et al. Electrospray ionization for large biomolecules. Science. 1989; 246:64 - 71.
[12] Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988; 60: 2299 - 2301
[13] Shen Y, et al. Nanoscale proteomics. Anal Bioanal Chem. 2004:378:1037 1045. [14] Link AJ, et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999;17:676 - 682.
[15] Crabb JW, Miyaji M, Gu X et al.Drusen proteome analysis: An approach to the etiology of age-related macular degeneration PNAS _ November 12, 2002 _ vol. 99 _ no. 23 _ 14683
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[20] Cavusoglu N, Thierse D. Differential Proteomic Analysis of the Mouse Retina. Molecular & Cellular Proteomics 2:494-505, 2003.
[21] Bresgen M, Baum U, Esser P, et al. Protein composition of the vitreous body in prolifertative diabetic retinopathy. An analysis with 2-D-electrophoresis. Ophthalmology, 1994, 91: 758-762
[22] Ouchi M, West K, Crabb JW. Proteomic analysis of vitreous from diabetic macular edema. Experimental Eye Research 81 (2005) 176 - 182
[23] Funatsu, H., Yamashita, H., Ikeda, T., Nakanishi, Y., Kitano, S., Hori, S., 2002. Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with diabetic macular edema and other retinal disorders. Am. J. Ophthalmol. 133, 537 - 543.
[24] Gao BB, Clermont A, Rook S. Extracellular carbonic anhydrase mediates he- morrhagic retinal and cerebral vascular permeability through prekallikrein ac- tivation.Nat Med. 2007 Feb;13(2):181-8. Epub 2007 Jan 28.
[25] Garcia RM, Canals F, Hernandez C. Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of Proliferative diabetic retinopathy. Diabetologia. 2007 Jun; 50(6): 1294-303. Epub 2007 Mar 23.
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18. Ward MM, Jobling Al, Kalloniatis M, Fletcher EL Glutamate uptake in retinal glial cells during diabetes. Diabetologia. 2005, 48: 351-360.
19. N. Adachi-Uehara et al. Up-regulation of genes for oxidative phosphorylation and protein turnover in diabetic mouse retina. Experimental Eye Research. 2006, 83:849-857.
20. Miyamoto K, Khosrof S, Bursell S-E, et al. Prevention of leukostasis and vascular leakage in streptozotocin-induced diabetic retinopathy via intercellular adhesion molecule-1 inhibition. Proc Natl Acad Sci USA. 1999, 96:10836-10841.
21. Zhang J, Gerhardinger C, Lorenzi M. Early complement activation and decreased levels of glycosylphosphatidylinositol-anchored complement inhibitors in human and experimental diabetic retinopathy.Diabetes. 2002, 51:3499-3504.
22. Rungger-Bra¨ndle E, Dosso AA, Leuenberger PM. Glial reactivity an early feature of diabetic retinopathy. Invest Ophthalmol Vis Sci. 2000, 41:1971-1980.
23. Carmo A, Cunha-Vaz JG, Carvalho AP, Lopes MC. L-arginine transport in retinas from strep-tozotocin diabetic rats: correlation with the level of IL-1_ and NO synthase activity. Vision Res. 1999, 39:3817-3823.
24. Gerhardinger C, Costa MB, Coulombe MC. Acute-Phase Response Proteins in Diabetic Retinopathy. IOVS. 2005, 46 (1)
25. King GL, Brownlee M, et al. The cellular and molecular mechanisms of diabetic complications. Endocrinol. Metab. Clin. N. Am. 1996, 25 :255-270.
26. Fields S. Proteomics in Genome land. Science. 2001,16(291): 1221-1224 [DOI: 10.1126/science.291.5507.1221]
27. Wasinger VC, Cordwell SJ, Cerpa-Poljak A, et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis. 1995,16:1090-1094
28. Anderson NG, Anderson NL, Human Proteins Index Project. Behring Inst. Mitt. 1979, 63: 169-210.
29. Kenyon G, et al. Defining the mandate of proteomics in the post-genomics era: workshop report.Mol Cell Proteomics. 2002,1:765-780.
30. O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975, 250:4007-4021.
31. Klose J. Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals. Humangenetik. 1975, 26:231-243.
32. Bjellqvist B, Ek K, Righetti PG, et al. Isoelectric focusing in immobilized pH gradients: principle, methodology and some applications. J Biochem Biophys Methods. 1982, 6: 317-339.
33. Grog A, Obermaier C, Boguth G, et al. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis. 2000, 21:1037-1053.
34. Corthals GL, et al. The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis. 2000, 21:1104-1115.
35. Washburn MP, Wolters D, Yates JR III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001,19:242-247.
36. Fenn JB, et al. Electrospray ionization for large biomolecules. Science.1989, 246:64-71.
37. Karas M, Hillenkamp F. Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal Chem. 1988, 60:2299-2301.
38. Shen Y, et al. Nanoscale proteomics. Anal Bioanal Chem. 2004, 378:1037-1045.
39. Link AJ, et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999, 17:676-682.
40. Bresgen M, Baum U,Esser P, et al. Protein composition of the vitreous body in prolifertative diabetic retinopathy. An analysis with 2-D-electrophoresis. Ophthalmology. 1994, 91: 758-762.
41. Ouchi M, West K, Crabb JW, et al. Proteomic analysis of vitreous from diabetic macular edema. Experimental Eye Research. 2005, 81:176-182.
42. Funatsu H, Yamashita H, Ikeda T, et al. Angiotensin II and vascular endothelial growth factor in the vitreous fluid of patients with diabetic macular edema and other retinal disorders. Am. J. Ophthalmol. 2002,133: 537-543.
43. Gao BB, Clermont A, Rook S, et al. Extracellular carbonic anhydrase mediates hemorrhagic retinal and cerebral vascular permeability through prekallikrein activation. Nat Med. 2007,13(2): 181-8.
44. Garcia RM, Canals F, Hernandez C, et al. Proteomic analysis of human vitreous fluid by fluorescence-based difference gel electrophoresis (DIGE): a new strategy for identifying potential candidates in the pathogenesis of Proliferative diabetic retinopathy. Diabetologia. 2007 Jun, 50(6):1294-303.
45. Quin GJ, Len AC, Billson FA, et al. Proteome map of normal rat retina and comparison with the proteome of diabetic rat retina: New insight in the pathogenesis of diabetic retinopathy. Proteomics. 2007, 7: 2636-2650.
46. Li Q, Puro DG, et al. Diabetes-induced dysfunction of the glutamate transporter in retinal Muller cells. IOVS. 2002, 43(9):3109-3116.
47. Lieth E, LaNoun KF, Antonetti DA, et al. Diabetes reduces glutamate oxidation and glutamine synthesis in the retina. Exp Eye Res. 2000, 70:723-730.
48. Liu Y, Huang MH, et al. Screening of the genes related to CNS development and injury by high density genechips. ACTA. 2004, 26(24)
49. Takagi C, Bursell SE, Lin YW, et al. Regulation of retinal hemodynamics in diabetic rats by increased expression and action of endothelin-1. IOVS.1996, 37:2504-2518.
50. Ishii H, Jirousek MR, Koya D, et al. Amelioration of vascular dysfunctions in diabetic rats by an oral PKCβ inhibitor. Science. 1996, 272:728-731.
51. Kim YH, Kim YS, et al. Expression of 14-3-3 zeta and interaction with protein kinase C in the rat retina in early diabetes. Diabetologia. 2005, 48(7):1411-1415.
52. Boyd-Kimball D, Castegna A, Sultana R, et al. Proteomic identification of proteins oxidized by Abeta (1-42) in synaptosomes: implications for Alzheimer's disease. Brain Res. 2005, 24, 1044(2):206-215.
53. LinksSultana R, Boyd-Kimball D, Poon HF, et al. Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD.Neurobiol Aging. 2006, 27 (11):1564-1576
54. Hong LE, Wonodi I, Avila M, et al. Dihydropyrimidinase-related protein 2 (DRP-2) gene and association to deficit and nondeficit schizophrenia. Am J Med Genet B Neuropsychiatr Genet. 2005,136(1):8-11.
55. Greene ND, Bamidele A, Choy M, et al. Proteome changes associated with hippocampal MRI abnormalities in the lithium pilocarpine-induced model of convulsive status epilepticus. Proteomics. 2007, 7(8): 1336-1344.
56. Perluigi M, Fai Poon H, Hensley K, et al. Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice-a model of familial amyo-trophic lateral sclerosis. Free Radic Biol Med. 2005,1, 38(7): 960-968.
57. Chen A, Liao WP, Lu Q, et al. Upregulation of dihydropyrimidinase-related protein 2, spec-trin alpha II chain, heat shock cognate protein 70 pseudogene 1 and tropomodulin 2 after focal cerebral ischemia in rats--a proteomics approach. Neurochem Int. 2007, 50(7-8): 1078-1086.
58. Nagelhus EA, Veruki ML, Torp R, et al. Aquaporin-4 water channel protein in the rat retina and optic nerve: polarized expression in Muller cells and fibrous astrocytes. J Neurosci. 1998,18: 2506-2519.
59. Hamann S, Zeuthen T, La Cour M, et al. Aquaporins in complex tissues: distribution of aquaporins 1-5 in human and rat eye. Am J Physiol. 1998, 274:1332-1345.
60. Manley GT, Fujimura M, Ma T, et al. Aquaporin-4 deletion in mice reduces brain edema following acute water intoxication and ischemic stroke. Nat Med. 2000, 6:159-163.
61. Newman EA. Regulation of extracellular and pH by polarized ion fluxes in glial cells: the retinal Muller cell. Neuroscientist. 1996, 2:1174-1175.
62. Nagelhus EA, Horio Y, Inanobe A, et al. Immunogold evidence suggests that coupling of K siphoning and water transport in rat retinal Muller cells is mediated by a coenrichment of Kir4.1 and AQP4 in specific membrane domains. Glia. 1999, 26:47-54.
63. Antcliff RJ, Marshall J, et al. The pathogenesis of edema in diabetic maculopathy. Semin Ophthalmol. 1999,14: 223-232.
64. Quagliarello VJ, Wispelwey B, Long Jr WJ, et al. Recombinant human interleukin-1 induces meningitis and blood-brain barrier injury in the rat: characterization and comparison with tumor necrosis factor. Journal of Clinical Investigation. 1991, 87(4): 1360-1366.
65. Kowluru RA, Odenbach S. Role of interleukin-1β in the pathogenesis of diabetic retinopathy. British Journal of Ophthalmology, 2004, 88(10): 1343-1347.
66. Du Y, Sarthy VP, Kern TS. Interaction between NO and COX pathways in retinal cells exposed to elevated glucose and retina of diabetic rats. American Journal of Physiology. 2004, 287: 735-741.
67. Vincent JA, Mohr S. Inhibition of caspasel/interleukin-1β signaling prevents degeneration of retinal capillaries in diabetes and galactosemia. Diabetes. 2007, 56(1): 224-230.
68 Ayalasomayajula SP, Amrite AC, Kompella UB, et al. Inhibition of cyclooxygenase-2, but not cyclooxygenase-1, reduces prostaglandin E2 secretion from diabetic rat retinas. European Journal of Pharmacology. 2004, 498(1-3): 275-278.
69. Ayalasomayajula SP, Kompellal UB. Celecoxib, a selective cyclooxygenase-2 inhibitor, inhibits retinal vascular endothelial growth factor expression and vascular leakage in a streptozoto-cin-induced diabetic rat model. European Journal of Pharmacology. 2003,458(3): 283-289.
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