慢性高眼压下兔视网膜组织蛋白质组变化的初步研究
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摘要
目的:本研究在建立兔慢性高眼压动物模型的基础上,采用双向电泳(two-dimensional gel electrophoresis , 2-DE)和液相色谱芯片串联质谱(LC-CHIP-MS/MS)分析技术,研究慢性高眼压下的视网膜组织蛋白质表达谱的变化,为青光眼视网膜神经节细胞(retinal ganglion cells , RGCs)的损伤机制和防治研究提供新的思路。
方法:1.动物模型的建立:实验动物左眼前房内注入0.2 ml复方卡波姆溶液制作成慢性高眼压模型,右眼为正常对照眼。慢性高眼压模型以眼压>22 mmHg并能持续1周为标准。如眼压<22 mmHg,7 d后按上述方法重复注药1次。造模成功后每周测2次眼压。在造模后28 d摘除动物眼球,取视网膜组织,提取视网膜组织蛋白质。
2. 2-DE:根据Bio-Rad双向电泳实验指南进行。2-DE的分离结果用经典质谱兼容银染法染色后进行图像扫描,以PDQest7.4.0图像分析软件分析凝胶并寻找差异点。根据差异分析结果,两种样品中的同一种蛋白质如存在2倍以上的光密度差异,则视为具有表达量的差异。
3.经LC-CHIP-MS/MS分析,得到差异蛋白的质谱图,用Spectrum Mill在UniProtKB/SWISS-PROT数据库及IPI-International Protein Index数据库中检索鉴定差异表达蛋白质,并进行生物信息学分析。
结果:1.慢性高眼压组兔眼压(intraocular pressure,IOP)术后3 h开始升高,术后14 d左右达到40 mmHg以上,之后大多数兔眼压维持在30～40 mmHg;正常对照组兔眼压在整个实验过程中无升高,平均为13～l5 mmHg。
2.慢性高眼压诱导视网膜组织9个蛋白点出现差异表达,其中3个蛋白点出现明显差异表达(均为上调)。LC-CHIP-MS/MS鉴定出3个蛋白质为:热休克蛋白70(heat shock 70 kD protein , HSP70),丙酮酸激酶(Pyruvate kinase)和烯醇酶(enolase)。
结论: 1.前房内注入复方卡波姆溶液是一种较理想的制作慢性高眼压动物模型的方法。
2.通过2-DE和LC-CHIP-MS/MS分析,发现兔视网膜蛋白质表达与对照眼相比有质和量的变化,这些变化涉及与RGCs糖酵解及应激反应有关的几组蛋白质,提示上述蛋白质组改变可能参与了慢性青光眼RGCs凋亡的过程。
Objective: Rabbits with chronic high intraocular pressure were establishhed in this study to investigate the effect of chronic high intraocular pressure on profile of retinal protein expression by two-dimensional electrophoresis (2-DE), and Liquid Chromatographic Chip Mass Spectrometry (LC-CHIP-MS/MS) to throw new light on mechanism research that how glaucoma injure to retinal ganglion cells (RGCs) and treatment.
Methods: 1. Animal model: 0.2 ml carbomer solution injection to the left anterior chamber of experimental animals to produce chronic high intraocular pressure model, whereas the right eye for the normal control. If the models keep intraocular pressure > 22 mmHg for one week, we take that for granted. If intraocular pressure <22 mmHg, procedure was repeated as above after 7 days. After the modeling, intraocular pressure was measured 2 times per week. Eyes of the model animals were removed after28 days of the construction, and protein of retinal tissue was extracted from retinal tissue.
2. 2-DE: Performed as guideline of Bio-Rad two-dimensional electrophoresis test. The results of 2-DE separation were performed image analysis after the classic silver staining compatible with MS, to detect difference by PDQest7.4.0 image analysis software. Two samples with the same protein that with more than 2 times of the optical density differences was considered as different of the expression.
3. By LC-CHIP-MS/MS analysis, mass spectrograms were developed. Differentially expressed proteins were searched in the UniProtKB/ SWISS-PROT and IPI-International Protein Index database using the Spectrum Mill to identify and access their biological information.
Results: 1. In the group of Chronic high intraocular pressure. intraocular pressure(IOP) began to increase after 3 h of construction, and reach 40 mmHg or more after about 14 d . Then most of the IOP remained at 30 ~ 40 mmHg. In normal control group, IOP maintain normal at an average of 13 ~ l5 mmHg throughout the experiment.
2. There are 9 proteins differentially expression, 3 of which were significant by up-regulated. LC-CHIP-MS/MS identified three proteins as: heat shock protein 70 (heat shock 70 kD protein, HSP70), pyruvate kinase and enolase.
Conclusion: 1. Carbomer solution injection to anterior chamber is an ideal means to construct animal models with chronic high intraocular pressure.
2. 2-DE and LC-CHIP-MS/MS analysis showed that compared with normal control eyes, the quality and quantity of expression of retinal proteins in chronic high intraocular pressure group changes which involve several groups of proteins related to RGCs glycolysis and stress response. Our findings indicate that these proteins may be involved in the process of RGCs apoptosis in chronic glaucoma.
方法:1.动物模型的建立:实验动物左眼前房内注入0.2 ml复方卡波姆溶液制作成慢性高眼压模型,右眼为正常对照眼。慢性高眼压模型以眼压>22 mmHg并能持续1周为标准。如眼压<22 mmHg,7 d后按上述方法重复注药1次。造模成功后每周测2次眼压。在造模后28 d摘除动物眼球,取视网膜组织,提取视网膜组织蛋白质。
2. 2-DE:根据Bio-Rad双向电泳实验指南进行。2-DE的分离结果用经典质谱兼容银染法染色后进行图像扫描,以PDQest7.4.0图像分析软件分析凝胶并寻找差异点。根据差异分析结果,两种样品中的同一种蛋白质如存在2倍以上的光密度差异,则视为具有表达量的差异。
3.经LC-CHIP-MS/MS分析,得到差异蛋白的质谱图,用Spectrum Mill在UniProtKB/SWISS-PROT数据库及IPI-International Protein Index数据库中检索鉴定差异表达蛋白质,并进行生物信息学分析。
结果:1.慢性高眼压组兔眼压(intraocular pressure,IOP)术后3 h开始升高,术后14 d左右达到40 mmHg以上,之后大多数兔眼压维持在30～40 mmHg;正常对照组兔眼压在整个实验过程中无升高,平均为13～l5 mmHg。
2.慢性高眼压诱导视网膜组织9个蛋白点出现差异表达,其中3个蛋白点出现明显差异表达(均为上调)。LC-CHIP-MS/MS鉴定出3个蛋白质为:热休克蛋白70(heat shock 70 kD protein , HSP70),丙酮酸激酶(Pyruvate kinase)和烯醇酶(enolase)。
结论: 1.前房内注入复方卡波姆溶液是一种较理想的制作慢性高眼压动物模型的方法。
2.通过2-DE和LC-CHIP-MS/MS分析,发现兔视网膜蛋白质表达与对照眼相比有质和量的变化,这些变化涉及与RGCs糖酵解及应激反应有关的几组蛋白质,提示上述蛋白质组改变可能参与了慢性青光眼RGCs凋亡的过程。
Objective: Rabbits with chronic high intraocular pressure were establishhed in this study to investigate the effect of chronic high intraocular pressure on profile of retinal protein expression by two-dimensional electrophoresis (2-DE), and Liquid Chromatographic Chip Mass Spectrometry (LC-CHIP-MS/MS) to throw new light on mechanism research that how glaucoma injure to retinal ganglion cells (RGCs) and treatment.
Methods: 1. Animal model: 0.2 ml carbomer solution injection to the left anterior chamber of experimental animals to produce chronic high intraocular pressure model, whereas the right eye for the normal control. If the models keep intraocular pressure > 22 mmHg for one week, we take that for granted. If intraocular pressure <22 mmHg, procedure was repeated as above after 7 days. After the modeling, intraocular pressure was measured 2 times per week. Eyes of the model animals were removed after28 days of the construction, and protein of retinal tissue was extracted from retinal tissue.
2. 2-DE: Performed as guideline of Bio-Rad two-dimensional electrophoresis test. The results of 2-DE separation were performed image analysis after the classic silver staining compatible with MS, to detect difference by PDQest7.4.0 image analysis software. Two samples with the same protein that with more than 2 times of the optical density differences was considered as different of the expression.
3. By LC-CHIP-MS/MS analysis, mass spectrograms were developed. Differentially expressed proteins were searched in the UniProtKB/ SWISS-PROT and IPI-International Protein Index database using the Spectrum Mill to identify and access their biological information.
Results: 1. In the group of Chronic high intraocular pressure. intraocular pressure(IOP) began to increase after 3 h of construction, and reach 40 mmHg or more after about 14 d . Then most of the IOP remained at 30 ~ 40 mmHg. In normal control group, IOP maintain normal at an average of 13 ~ l5 mmHg throughout the experiment.
2. There are 9 proteins differentially expression, 3 of which were significant by up-regulated. LC-CHIP-MS/MS identified three proteins as: heat shock protein 70 (heat shock 70 kD protein, HSP70), pyruvate kinase and enolase.
Conclusion: 1. Carbomer solution injection to anterior chamber is an ideal means to construct animal models with chronic high intraocular pressure.
2. 2-DE and LC-CHIP-MS/MS analysis showed that compared with normal control eyes, the quality and quantity of expression of retinal proteins in chronic high intraocular pressure group changes which involve several groups of proteins related to RGCs glycolysis and stress response. Our findings indicate that these proteins may be involved in the process of RGCs apoptosis in chronic glaucoma.
引文
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[2] Foster PJ.Johnson GJ . Glaucoma in China:how big is the problem?Br J Ophthalmo1.2001,85:1277-1282.
[3]胡铮,赵家良,董方田,等.北京市顺义县青光眼流行病学调查.中华眼科杂志. 1989,25:ll5-ll9.
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[5] Sim DHJ,Goh LG,Ho T.Glaucoma pattern amongst the elderly Chinese in Singapore.Ann Acad Med Singapore.1998,27:819-823.
[6] Foster PJ, Oen FTS, Machin D, et al. The prevalence of glaucoma in Chinese residents of Singapore.Arch Ophthalmo1.2000,118:1105-1111.
[7] Frezzotyi R,Renieri A,Frezzoth P.Adult-onset primary glaucoma and molecular genetics:a review[J]. Eur J Ophthalmol.2004,14(3):220-225.
[8] ValentováJ.Blockers of beta-adrenergic receptors--a group of chiral agents stereoselective synthesis of beta-blockers. Ceska Slov Farm. 2005,9(5): 201-206
[9] Jeffcry DA,Bogyo M.Chemical proteomics and its application to drug discovery [J]. Curr Opin Bioteehno1.2003,14(1):87-95.
[10] Emi Nakajima, Larry L D, Cory Bystrom, et al. Calpain-Specific Proteolysis in Primate Retina: Contribution of Calpains in Cell Death[J]. Invest Ophthalmol Vis Sci. 2006:47(11):5469-5475.
[11] Bhattacharya S K, Crabb J S, Bonilha V L, et al.Proteomics Implicates Peptidyl Arginine Deiminase 2 and Optic Nerve Citrullination in Glaucoma Pathogenesis Invest[J]. Ophthalmol. Vis Sci.2006,47(7):2508–2514.
[12] Picciani R G, Diaz A,Lee R K, et al.Potential for Transcriptional Upregulation of Cochlin in Glaucomatous Trabecular Meshwork: A Combinatorial Bioinformatic and Biochemical Analytical Approach Invest[J]. Ophthalmol Vis Sci.2009,50 (7): 3106-3111.
[13] Boehm N, Thiel U, Lossbrandt U, et al. Intraindividual Comparison of Antibody Patterns in Sera and Aqueous Humor of Glaucoma Patients and Healthy Subjects. Invest[J]. Ophthalmol. Vis Sci, 2009,50:2076.
[14] Kruger N J. The Brandford method for protrin quantitation[J].Methods Mol Biol.1994,32(1):9-15.
[15] Sears D,Sear M.Blood-aqueous barrier and alpha-chymotrypsina glaucoma in rabbits.Am J Ophthamol.1974,77:378-383.
[16] Lessell S,Knwabara T, Experimental alpha-chymotrypsina glaucoma Arch Ophthamol.1969,81;853-864.
[17] Zhu MD,Cai FY.Development of experimental chronic intraocular hytertension in the rabbit.Aust N Z J Ophthamol.1992,20;225-234.
[18] Wood DC,Contaxis I,Sweet D,et al.Response of rabbits to conticosteroids: I, influence on growth,intraocular pressure and lens transparency.Am J Ophthamol, 1967, 63; 841 -849.
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