论文标题:增生性玻璃体视网膜病变的玻璃体蛋白质组及其分子标志物的研究
Study on Vitreous Proteomic Analysis and Biomarker Discovery of Proliferative Vitreoretinopathy
论文作者
论文导师 王方;吴星伟;樊莹;邱庆华,论文学位 博士,论文专业 眼科学
论文单位 上海交通大学,点击次数 107,论文页数 148页File Size1902K
2008-03-01论文网 http://www.lw23.com/lunwen_108809072/
Proliferative vitreoretinopathy;; proteome;; vitreous;; biomarker;; liquid chromatography;; two-dimensional gel electrophoresis
目的增生性玻璃体视网膜病变(proliferative vitreoretinopathy, PVR)是导致视网膜脱离手术失败最主要的原因。本研究利用蛋白质组学平台,对原发性孔源性视网膜脱离伴PVR的玻璃体蛋白质组进行分离和鉴定,建立PVR和正常眼玻璃体蛋白质组图谱,筛选能评估术前PVR严重程度和手术预后的血清分子标志物。 方法(1)二维液相色谱联合串联质谱(two-dimensional-liquid chromatography coupled by tandem mass spectrometry, 2D-LC-MS/MS)分离和鉴定PVR玻璃体的蛋白质组:收集PVRB、C、D级患者的玻璃体原液和术前血清,正常捐献眼玻璃体和健康人的血清为对照组。将相同等级的轻度PVR(PVRB级)和严重PVR(PVRC、D级)以及捐献眼玻璃体样本等量混合,丙酮沉淀过夜。重溶后测蛋白质浓度,加入胰酶,37℃酶解过夜。过阳离子交换柱后进行在线反向色谱,纳喷雾质谱鉴定,MASCOT数据库检索。( 2 )双向凝胶电泳( two-dimensional gel electrophoresis,2-DE)联合基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption/ ionization - time of flight-mass spectrometry,MALDI-TOF-MS)对PVR与正常玻璃体的差异蛋白质组研究:实验样本同(1)。将同一等级的玻璃体样本等量混合,丙酮沉淀过夜。再水化液重溶后,加至固相pH梯度凝胶电泳胶条(18 cm,pH3-10)进行水化,等电聚焦,100 V/30 min,3500 V/ 4 h,8000 V/4 h。平衡后进行聚丙烯酰胺凝胶电泳,30 mA / 6 h。银染后图像分析。差异明显(至少2倍以上的吸光度OD)的蛋白质点进行胶内酶解,质谱鉴定后MASCOT数据库检索。(3)激肽原1在PVR玻璃体和血清中的验证:PVR B、C、D级和正常捐献眼玻璃体样本和相应的血清样本同(1)。同时,收集PVR A级的玻璃体和术前血清样本以及PVR术后不同状态的血清样本,包括环扎术后未愈者和硅油眼。对玻璃体和相应的血清样本分别进行激肽原1的免疫印记分析(western blotting analysis, WB)和酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)。(4)类胰岛素生长因子结合蛋白-6 (insulin-like growth factor binding protein,IGFBP-6)在PVR的玻璃体和血清中的验证:实验样本同(3)。应用WB和ELISA对玻璃体和相应的血清样本进行验证。 结果(1)在捐献眼、轻度和严重PVR玻璃体中分别鉴定到129、97和137种蛋白质。其中,35种为三者共有的蛋白质,24种为PVR玻璃体特异蛋白质。血浆中的高丰度蛋白质,如丝氨酸蛋白酶抑制剂家族、补体成分等和一些细胞外蛋白质在PVR玻璃体中明显上调或增加;同时细胞骨架蛋白质微管蛋白以及参与糖酵解的酶,如丙酮酸激酶和烯醇化酶等在PVR中明显下调。在PVR特异的蛋白质中,激肽原1是鉴定到的肽段数较多大的相对高丰度蛋白质;而IGFBP-6与增殖的信号转导有关。(2)捐献眼、轻度PVR和严重PVR玻璃体2-DE图谱分别分析到47、184和336个蛋白质点,在13个明显差异蛋白质中成功的鉴定到7种蛋白质。其中,烯醇酶2是捐献眼特有的;甲状腺激素结合蛋白单体和视黄醇结合蛋白(retinol-binding protein,RBP)单链是在严重PVR玻璃体中特异的蛋白质;前列腺素D合成酶和RBP3前体是捐献眼和PVR眼玻璃体共有的蛋白质,轻度PVR时明显增加,严重PVR时下调。血清白蛋白和转铁蛋白,随PVR严重程度增加而明显上调。(3)WB显示激肽原1在PVR患者玻璃体和血清中检测到而在正常捐献眼和正常人的血清中未测到,且在PVRC、D级中明显高于PVRB级。激肽原1可以在所有的PVR患者玻璃体和血清样本中检测到(24/24, 100%)。ELISA显示严重PVR患者玻璃体和血清中激肽原1明显高于轻度PVR(P<0.05)。PVR患者术后6个月血清中激肽原1明显下降(P<0.05),仍高于正常对照组(P<0.05)。但环扎术后未愈者血清中激肽原1明显高于正常对照组和硅油眼(P<0.01),亦高于玻切术后6个月随访组(P<0.05)。(4)WB显示,IGFBP-6在PVR的玻璃体和血清中检测到而在正常捐献眼和正常人血清中未测到,且在PVRC、D级中明显高于PVRB级。IGFBP-6可以在91.7%的PVR患者玻璃体和血清样本中检测到(除外2个PVRB样本)。ELISA显示严重PVR患者的玻璃体和血清中IGFBP-6明显高于轻度PVR(P<0.05)。PVR患者术后6个月血清中IGFBP-6的浓度明显下降(P<0.05),与玻璃体术后硅油眼和正常对照组的血清中的浓度接近(P>0.05),但明显低于环扎术后未愈组(P<0.05)。 结论(1)本研究提示PVR是一个复杂的病理过程,玻璃体发生一系列的变化,有大量的新生蛋白质生成、血浆蛋白质的侵入以及正常玻璃体的蛋白质降解,证实了PVR的病程中存在血-视网膜屏障的破坏、细胞骨架的重塑、细胞免疫反应参与以及玻璃体的能量代谢障碍。(2)2D-LC-MS/MS和2-DE-MALDI-TOF-MS蛋白质策略的联合应用可以互为补充,能够更完整的揭示差异蛋白质组的研究(。3)激肽原1和IGFBP-6是候选的PVR血清分子标志物,可用于PVR严重程度的临床分级和预后评价指标,并为预防PVR的药物开发提供了新的靶向,有一定的临床应用前景。
Purpose: Proliferative vitreoretinopathy (PVR) is the most common cause of anatomic failure in retinal detachment surgery. To understand the molecular mechanisms, vitreous proteomes of rhegmatogenous retinal detachment patients with PVR were investigated by proteomic strategies. The purpose of the study is to separate and identify the vitreous proteome of PVR and donor eyes in order to fish for the serum biomarker which can evaluate the severity of PVR preoperatively and prognosis postoperatively. Methods: (1) To separate and identify the vitreous proteome of PVR by two-dimensional-liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS): Vitreous samples of moderate PVR (grade B), and severe PVR (grade C or D) were aspirated during pars plana vitrectomy (PPV) before infusion and the serum of those patients were collected preoperatively. The normal vitreous from donor eyes and normal serum from healthy humans was as the control. The vitreous samples in the same group were mixed at same volume before the experiment. Cold acetone was added into the vitreous samples overnight. The samples were lysed in Tris base buffer, and then were digested into peptides using sequencing grade trypsin overnight at 37°C. The protein mixtures from the samples were digested and then fractionated into 10 subgroups by strong cation exchange (SCX) chromatography. The peptide mixtures of each SCX fraction were then loaded onto a reverse phase (RP) trap column for desalting. A spray voltage of 2500V was applied to a PicoTip nanospray emitter (New Objective) to give a steady spray. The MS/MS spectras were searched against the human protein databases using MASCOT. (2)The differential vitreous proteome between PVR and donor by two-dimensional gel electrophoresis coupled with matrix assisted laser desorption / ionization - time of flight - mass spectrometry (2-DE-MALDI-TOF-MS) : The samples were same as the first part. The vitreous samples in the same group were mixed at same volume before the experiment. Cold acetone was added into the vitreous samples overnight. First dimension isoelectric 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 then IEF was performed at 100 V for 30 min, linear increase to 3500 V for 4 hr, and then held at 8000 V for 4 hr. The second dimension 12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) was performed at 30 mA for 6 hr after the IPG strips were equilibrated. After silver staining, the gels were analyzed by the 2-DE gel analysis software. The matched spots of which exhibited at least two-fold difference in average optical density (OD) % were excised and tryptic digested in-gel. The pepetides were analyzed by MALDI-TOF-MS and the MS/MS spectras were searched against the human protein databases using MASCOT. (3) To verify kininogen 1 in the vitreous and serum samples. The vitreous of the donor and PVRB, C, D and the corresponding serum were same as the first part. Furthermore, the vitreous of PVR A and the corresponding serum preoperatively was collected. The serum samples from the patients which silicon oil were tamponaded in PPV (group SO) or were uncured after scleral buckling surgery (group SBS) were collected. All the vitreous and serum samples were performed western blotting analysis (WB) and enzyme linked immunosorbent assay (ELISA). (4) To verify insulin-like growth factor binding protein (IGFBP-6)in the vitreous and serum samples. The samples were same as the third part. To verify IGFBP-6, all the vitreous and serum samples were performed WB and ELISA. Results: (1) In the current study, 129, 97 and 137 proteins were identified in vitreous of donor, moderate and severe PVR, including 35 overlap proteins and 24 PVR special proteins. In PVR vitreous samples, complement components, serine proteinase inhibitors, and extracellular proteins were up-regulated or increased, while normal cytoskeleton and enzymes involved in glycolysis, such as pyruvate kinase and enolases, were down-regulated or disappeared. Among the PVR special proteins, kininogen 1 was one of the relatively high abundance proteins of which more peptides could be identified; IGFBP-6 was involved in signal transduction. (2) There were 47, 184 and 336 protein spots were analyzed from the 2-DE gels of donor, moderate PVR and severe PVR. Among 13 the significantly difference spots, 7 proteins were successfully identified. Enolase 2 was the special protein in donor vitreous; transthyretin monomer and retinol-binding protein (RBP) chain B were special proteins in severe PVR vitreous; prostaglandin D synthase and RBP3 precursor were the overlap proteins,which were upregulated in moderate PVR but downregulated in severe PVR; albumin and transferrin were upregulated associated with the severity of PVR. (3) WB outcomes presented that kininogen 1 was detected in both vitreous and the corresponding serum of PVR patients but was not detected in either donor vitreous or normal serum. Kininogen 1 expressed highly in the vitreous and serum of PVR C and D than those in PVRB. Kininogen 1 could be detected in 100% of vitreous and serum samples. The ELISA outcomes indicated that the concentration of kininogen 1 in vitreous and serum samples of severe PVR was significantly higher than in moderate PVR(P<0.05). Kininogen 1 was reduced significantly at 6 month postoperatively(P<0.05), but was still higher than normal (P<0.05). However, kininogen 1 in group SBS was significantly higher than in group SO and normal control (P<0.01), and was still higher than in 6 month postoperative followed-up group(P<0.05). (4) WB outcomes presented that IGFBP-6 was detected in both vitreous and the corresponding serum of PVR patients but was not detected in either donor vitreous or normal serum. The IGFBP-6 expressed highly in the vitreous and serum of PVR C and D than those in PVRB. IGFBP-6 could be detected in 91.7% of vitreous and serum samples except for 2 samples of PVRB. The ELISA outcomes indicated that the concentration of IGFBP-6 in vitreous and serum samples of severe PVR was significantly higher than in moderate PVR(P<0.05). IGFBP-6 was reduced significantly at 6 month postoperatively(P<0.05), and no significant difference was found comparing with the normal and group SO(P>0.05). However, IGFBP-6 in group SBS was significantly higher than in normal, group SO and postoperative group(P<0.05). Conclusions: (1) Out current proteome study presented that PVR was a complicated pathology process with great amount of proteins newly produced, serum proteins invaded in and normal proteins reduced. These outcomes indicated that destruction of blood-retinal barrier, metabolism dysfunction, immune reactions, and cytoskeleton remolding were all involved in the process of PVR. (2) The combination of 2D-LC-MS/MS and 2-DE-MALDI-TOF-MS could be complemented to display the integrated differential proteome. (3) Kininogen 1 and IGFBP-6 may be candidate serum biomarkers of PVR, which can evaluate the severity of PVR and preoperatively and prognosis postoperatively. Further investigations into these special proteins will provide additional targets for treatment or prevention of PVR. Our study provided exciting future in clinical practice
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