兔增殖性玻璃体视网膜病变视网膜蛋白质组学的初步研究
发布时间：2018-08-10 21:44
【摘要】：目的:( 1 )建立兔增殖性玻璃体视网膜病变(proliferative vitreoretinopathy , PVR)动物模型;( 2 )采用双向凝胶电泳(two-dimensional gel electrophoresis,2-DE)/高效液相色谱芯片串联质谱(high-performance liquid chromatography and tandem mass spectrometry using a chip cube nano-flow system ,HPLC-Chip MS/MS system)和Western blot方法筛选、鉴定PVR兔视网膜与正常兔表达有差异的蛋白,为探讨PVR的发病机制寻找新的突破口。 方法:(1)采用部分玻璃体切除+造成裂孔和视网膜脱离后饲养8周的方法建立PVR动物模型;(2)获取实验组和对照组视网膜标本并用clean-up kit处理视网膜标本;(3)运用2-DE对视网膜蛋白进行二维分离;(4)采集凝胶图像,以PDQuest软件进行图像分析,寻找有意义的差异蛋白点;(5)将差异蛋白质点挖出、酶解,进行HPLC-Chip MS/MS鉴定;( 6 )使用Mascot软件在UniProtKB/SWISS-PORT数据库中搜索鉴定差异表达蛋白,生物信息学分析,得到差异蛋白的相关肽段的MS/MS质谱图;(7)对鉴定出的、有意义的差异蛋白aB-晶状体蛋白运用Western blot方法进行一步验证。 结果:(1)八周后,行部分玻璃体切除+造成裂孔和视网膜脱离的18只眼中,有3只眼诱导出了PVR B级, 3只眼诱导出了PVR C级。对照组中6只眼无一只眼诱导出PVR;(2)运用2-DE分离出PVR与正常兔视网膜的差异蛋白点,经PDQuest8.0软件进行分析后,筛选10个表达有差异的蛋白点,运用HPLC-Chip MS/MS进行分析鉴定,发现有8个蛋白在PVR组表达上调。其中满足①肽评分8;②SPI(Structure predictability index for protein sequences) (%)70%;③分子量及等电点与双向电泳的结果相符的蛋白有2个,即aB-晶状体蛋白(Alpha-crystallin B chain - Oryctolagus cuniculus)和aA-晶状体蛋白(Alpha-crystallin A chain - Oryctolagus cuniculus)。(3)Western blot显示,aB-晶状体蛋白在PVR B、C级的视网膜中检测到,而在正常对照视网膜中未测到。 结论:aB-晶状体蛋白为PVR的潜在生物标志物,可能在PVR的发生、发展中起着重要作用。
[Abstract]:Objective: (1) to establish (proliferative vitreoretinopathy, PVR) animal model of proliferative vitreoretinopathy in rabbits; (2) to screen by two-dimensional gel electrophoresis2-DE / high-performance liquid chromatography and tandem mass spectrometry using a chip cube nano-flow system HPLC-Chip MS/MS system and Western blot method. To identify the differentially expressed proteins in the retina of PVR rabbits and normal rabbits, and to find a new breakthrough in the pathogenesis of PVR. Methods: (1) PVR animal model was established by partial vitrectomy and retinal detachment for 8 weeks; (2) retinal specimens of experimental group and control group were obtained and treated with clean-up kit; (3) Retinal specimens were treated with 2-DE. Omentum protein was separated by two-dimensional method. (4) Gel images were collected. PDQuest software was used for image analysis to find meaningful differential protein spots; (5) the differential protein spots were extracted, hydrolyzed and identified by HPLC-Chip MS/MS; (6) Mascot software was used to search and identify differentially expressed proteins in UniProtKB/SWISS-PORT database, and bioinformatics analysis was used to identify the differentially expressed proteins. The MS/MS mass spectra of the related peptides of differentially expressed proteins were obtained. (7) A Western blot method was used to verify the identified and meaningful differentially expressed proteins. Results: (1) in 18 eyes with partial vitrectomy, PVR B grade was induced in 3 eyes and PVR C grade was induced in 3 eyes after partial vitrectomy. In 6 eyes of the control group, none of the eyes induced PVR. (2) the differential protein spots of PVR and normal rabbit retina were isolated by 2-DE. Ten differentially expressed protein spots were screened by PDQuest8.0 software and identified by HPLC-Chip MS/MS. Eight proteins were found to be up-regulated in PVR group. Among them, there are 2 proteins that meet the 1 peptide score of 8 ~ 2 SPI (Structure predictability index for protein sequences) (%) 70 ~ (-3) molecular weight and isoelectric point, which are consistent with the results of two-dimensional electrophoresis. Alpha-crystallin B chain-Oryctolagus cuniculus) and Alpha-crystallin A chain-Oryctolagus cuniculus). (3) Western blot were detected in the retina of PVR B C grade, but not in the normal control retina. Conclusion as a potential biomarker of PVR, the PVR may play an important role in the pathogenesis and development of PVR.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R776.4;R774.1
本文编号：2176299
[Abstract]:Objective: (1) to establish (proliferative vitreoretinopathy, PVR) animal model of proliferative vitreoretinopathy in rabbits; (2) to screen by two-dimensional gel electrophoresis2-DE / high-performance liquid chromatography and tandem mass spectrometry using a chip cube nano-flow system HPLC-Chip MS/MS system and Western blot method. To identify the differentially expressed proteins in the retina of PVR rabbits and normal rabbits, and to find a new breakthrough in the pathogenesis of PVR. Methods: (1) PVR animal model was established by partial vitrectomy and retinal detachment for 8 weeks; (2) retinal specimens of experimental group and control group were obtained and treated with clean-up kit; (3) Retinal specimens were treated with 2-DE. Omentum protein was separated by two-dimensional method. (4) Gel images were collected. PDQuest software was used for image analysis to find meaningful differential protein spots; (5) the differential protein spots were extracted, hydrolyzed and identified by HPLC-Chip MS/MS; (6) Mascot software was used to search and identify differentially expressed proteins in UniProtKB/SWISS-PORT database, and bioinformatics analysis was used to identify the differentially expressed proteins. The MS/MS mass spectra of the related peptides of differentially expressed proteins were obtained. (7) A Western blot method was used to verify the identified and meaningful differentially expressed proteins. Results: (1) in 18 eyes with partial vitrectomy, PVR B grade was induced in 3 eyes and PVR C grade was induced in 3 eyes after partial vitrectomy. In 6 eyes of the control group, none of the eyes induced PVR. (2) the differential protein spots of PVR and normal rabbit retina were isolated by 2-DE. Ten differentially expressed protein spots were screened by PDQuest8.0 software and identified by HPLC-Chip MS/MS. Eight proteins were found to be up-regulated in PVR group. Among them, there are 2 proteins that meet the 1 peptide score of 8 ~ 2 SPI (Structure predictability index for protein sequences) (%) 70 ~ (-3) molecular weight and isoelectric point, which are consistent with the results of two-dimensional electrophoresis. Alpha-crystallin B chain-Oryctolagus cuniculus) and Alpha-crystallin A chain-Oryctolagus cuniculus). (3) Western blot were detected in the retina of PVR B C grade, but not in the normal control retina. Conclusion as a potential biomarker of PVR, the PVR may play an important role in the pathogenesis and development of PVR.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R776.4;R774.1
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