增生性玻璃体视网膜病变眼玻璃体中差异蛋白质组的鉴定

Differentiation of vitreous proteome in human eye with proliferative vitreoretinopathy

背景增生性玻璃体视网膜病变（PVR）是导致视网膜脱离手术失败的最主要原因之一。蛋白质组学是研究人体组织中差异蛋白的重要方法。目的应用双向凝胶电泳（2-DE）联合质谱分析法对原发性视网膜脱离伴PVR患者进行差异蛋白质组分析，筛选出PVR眼特异的蛋白质表达。方法经玻璃体切割术收集PVRB级（轻度PVR），PVRC、D级（重度PVR）患者各8例8眼的玻璃体标本进行（2-DE）联合质谱检测分析，正常供体8眼作为对照。抽取的玻璃体标本再水化液重溶后，加至固相pH梯度胶条（18CIn，pH3—10）水化，等电聚焦，平衡后进行聚丙烯酰胺凝胶电泳，银染。对差异明显的蛋白质点（至少2倍以上的吸光度）进行胶内酶解，质谱鉴定。结果供体眼、轻度PVR眼和严重PVR眼经2-DE图谱分别检测到玻璃体标本中47、184和336个蛋白质点，PVR增加的蛋白质点的pH等电点以5～8居多，轻度PVR和重度PVR分别为104（56．5％）和230点（68．5％），在13个明显差异点中鉴定到7种蛋白质。其中，烯醇酶为供体眼所特有，甲状腺激素结合蛋白单体和视黄醇结合蛋白（RBP）单链为重度PVR玻璃体中特异表达的蛋白质，前列腺素D合成酶和RBP3前体在供体眼和PVR眼玻璃体均能检测到，且在轻度PVR眼中表达量较高，严重PVR眼下调。玻璃体中血清白蛋白和转铁蛋白随PVR严重程度的增加而明显上调。结论PVR发病过程中，原有的玻璃体蛋白质发生降解，部分血清中的蛋白质明显k调，表明PVR眼有血一视网膜屏障的破坏。

Background Proliferative vitreoretinopathy （PVR） is a a common cause of anatomic failure in retinal detachment surgery. Proteomies is the critical method to investigate the different proteins. Objective This study was to search for related vitreous proteins in rhegmatogenous retinal detachment patients with proliferative vitreoretinopathy （PVR） and screen for the related protein expression in the vitreous with PVR. Methods Vitreous samples were obtained from 8 moderate PVR （grade B） eyes and 8 severe PVR （grade C or D） eyes during pars plana vitrectomy （PPV） for the proteomics analysis by two-dimensional gel electrophoresis cuupled with mass spectrometry. Normal vitreous from 8 healthy donor eyes served as control. First dimension isoelectrie focusing （IEF） was performed with the Pharmacia IPGphor in solvent B using 18 cm non-linear pH 3-10 immobilized pH gradient （IPG） strips. IPG strips were rehydrated with sample, and then IEF was performed. The second dimension 12% sodium dodecyl sulfate （SDS） polyacrylamide gel eleetropboresis （PAGE） was performed after the IPG strips were equilibrated. After silver staining,the gels were analyzed by the 2-DE gel analysis software. The matched spots were excised and tryptica digested in-gel. The peptides were analyzed by MALDI-TOF-MS and the MS/MS spectral were searched against the human protein databases using MASCOT. This study process complied with the Declaration ofHelsinki. Written informed consent was obtained from each patient prior to this trial. Results In the current study, a total of 47,184 and 336 protein spots were detected from the normal donor eyes, moderate PVR eyes and severe PVR eyes,respectively, by 2-DE gels. Among 13 protein spots with significant differences in the 3 groups, 7 types of proteins were successfully identified. The results indicated that Enolase 2 was a specific protein for normal donor vitreous,whereas transthyretin monomer and retinol-binding protein （RBP） chain B were distinct proteins found in severe PVR vitreous, and prostaglandin D synthase and RBP3 precursor were common proteins with up-regulated expression in vitreous samples with moderate PVR,hut down-regulated levels in vitreous samples with severe PVR. The levels of albumin and transferrin in vitreous showed dynamic elevation with the enhancement of severity of PVR. Conclusion The changes in the vitreous proteome imply that some types of vitreous proteins degrade and some common serum proteins are expressed in the vitreous body with PVR, which indicated the disruption of the blood- retinal barrier in the pathogenesis of PVR.