王方 于靖 刘锋 崔书建 宋正宇 应海燕 李慧 邱庆华 韩泽广
Department of Ophthalmology， Shanghai First People’s Hospital， Jiao-tong University， Shanghai 200080
Purpose： To separate and identify the vitreous proteome of PVR in order to fish for the serum biomarkers which can evaluate the severity of PVR preoperatively and prognosis postoperatively.
Method： The vitreous proteomes of rhegmatogenous retinal detachment patients with PVR were investigated 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 serum samples from the patients whose silicon oil (group SO) was tamponaded into the eyes in PPV and failure after scleral buckling surgery (group SBS) were collected. 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. All the vitreous and serum samples were performed western blotting analysis (WB) and enzyme linked immunosorbent assay (ELISA) in order to verify the candidate biomarkers.
Result： 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 which identified more peptides； insulin-like growth factor binding protein (IGFBP-6) was involved in signal transduction. The WB outcomes presented that kininogen 1 and IGFBP-6 were detected in both vitreous and the corresponding serum of PVR patients but were not detected in either donor vitreous or normal serum. Kininogen 1 and IGFBP-6 expressed highly in the vitreous and serum in PVR C and D than those in PVRB. Furthermore， kininogen 1 could be detected in 100% of vitreous and serum samples while 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 kininogen 1 and IGFBP-6 in vitreous and serum samples of severe PVR was significantly higher than in moderate PVR(P<0.05). Kininogen 1 and IGFBP-6 were reduced significantly at 6 month postoperatively(P<0.05)， but kininogen 1 was still higher than normal (P<0.05). However， kininogen 1 and IGFBP-6 in group SBS were significantly higher than in group SO and normal control (P<0.01)， and were still higher than in postoperative group(P<0.05).
Conclusions： Out current proteome study presented that PVR was a complicated pathology process with great amount of proteins newly produced. Kininogen 1 and IGFBP-6 may be the 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.
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