慢性高眼压下兔视网膜组织蛋白质组变化的初步研究

发布时间：2018-06-01 03:18

本文选题：慢性 + 高眼压　；参考：《重庆医科大学》2010年硕士论文

【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。
[Abstract]:Objective: to study the changes of protein expression profile of retinal tissue under chronic intraocular pressure (IOP) by using two-dimensional gel electrophoresis, 2-DEE and LC-CHIP-MS / MS / MS analysis technique in rabbits with chronic intraocular hypertension. To provide a new idea for the study of the damage mechanism and prevention and treatment of retinal ganglion cell (ganglion cells, RGCs) in glaucoma. Method 1: 1. Establishment of animal model: 0. 2 ml compound carbomer solution was injected into the left anterior chamber of experimental animals to make the model of chronic high intraocular pressure, and the right eye was a normal eye. The model of chronic high intraocular pressure was based on IOP 22 mmHg and lasted for 1 week. If the intraocular pressure was 22 mm HgG for 7 days, the drug was injected again according to the above method. IOP was measured twice a week after successful modeling. The animal eyeballs were removed and retina tissues were removed at 28 days after model making, and the protein of retinal tissue was extracted. 2. 2-DE: according to the Bio-Rad two-dimensional electrophoresis experimental guide, the separation of .2-DE was carried out by classical mass spectrometry compatible silver staining method. The gel was analyzed by PDQest7.4.0 image analysis software and the difference point was found. According to the results of differential analysis, if there is a difference in optical density of more than 2 times of the same protein in the two kinds of samples, it is regarded as having the difference of expression quantity. 3. The differential protein mass spectrogram was obtained by LC-CHIP-MS/MS analysis. Spectrum Mill was used to search and identify differentially expressed proteins in UniProtKB/SWISS-PROT database and IPI-International Protein Index database, and bioinformatics analysis was carried out. The result is 1: 1. In chronic high intraocular pressure group, intraocular pressure (IOP) began to increase at 3 h after operation and reached more than 40 mmHg on the 14th day after operation. After that, IOP was maintained at 30 ~ 40 mm Hg in most rabbits, while in normal control group, IOP did not increase during the whole experiment, with an average of 13~l5 Hg. 2. The differential expression of 9 protein spots in retinal tissue was induced by chronic intraocular pressure. Three of them were identified as heat shock protein (70(heat shock 70 kD protein, HSP70), pyruvate kinase (Pyruvate kinase) and enolase (enolase). Conclusion: 1. Injection of compound carbomer solution into anterior chamber is an ideal method for making chronic intraocular hypertension animal model. 2. By 2-DE and LC-CHIP-MS/MS analysis, the changes of protein expression in rabbit retina were found to be related to several groups of proteins related to RGCs glycolysis and stress reaction. These proteome changes may be involved in the process of RGCs apoptosis in chronic glaucoma.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R775.9

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