Screening And Identification Of Abnormal Expression Proteins From Retina-Choroid Complex In CNV Rat And Hypoxia Human RPE Cell In Vitro

[Background and Aims] Age-related macular degeneration (AMD) is one of the principal cause of registered legal blindness among the elderly. Although had carried out many researchs, we cannot know the exact etiopathogenisis and cannot find the wonderful treatment plan. In order to assess comparative protein expression, proteins extracted from whole retina and choroid of normal/CNV rat and normal/hypoxia human RPE cell in vitro were resolved by proteome approach. This study was aimed to Screening and identification of abnormal expression proteins from retina-choroid complex in CNV rat and hypoxia human RPE in vitro.[Materials and Methods] 8 BN rats, pupil dilated and anesthetized, were selected one eye randomly for test, and the other for control. Krypton laser (647 ran) run 20 spots photocoagulation in retina of the test eye. After 4 weeks, fundus fluorescein angiographic (FFA) played to affirm that the choroidal neovascularization is formed. And than, animals were executed, eye cups were made pathological section. Protein of retina-choroid complex in normal/CNV rats and Normal/ hypoxia human RPE cells, with exponential phase of growth, was extracted, and frozen by liquid nitrogen for proteome study. All sample performed isoelectric focusing electrophoresis (IEF), separated by isoelectric point (pI); progressed sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) and separated by protein molecular weight (Mr). 2D-gels revealed by Coomassie brilliant blue, analyzed by Image Master 2D Elite. Differential proteins spots were screened in normal/CNV retina-choroidcomplex and normal/hypoxia RPE cell. For identification of differential proteins, peptide masses were analyzed by matrix-assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF/MS).[Results] All of photocoagulated eyes had induced CNV. Comparison of 2D-spots that approximately 405 protein spots (matching rate within group was 90.26%) obtained in normal rat group, 436 spots (91.63%) in CNV rat group, and the average matching rate between groups was 89.22%. All together 44 spots changed signifficantly (Volume value ≥ 2.0 times), among them, 21 protein spots increased in abundance and 23 showed lower expression. 9 protein spots which changed obviously were chosen randomly to be performed mass spectrometry(MS) analysis, and 7 of them were identified successfully: eIF-5A, mitochondrial ATP synthase D chain, Cu, Zn-SOD, Endoplasmic retuclum protein 29(ERp29), and Transthyretin Chain D(TTR) were up-regulated. While μ-Crystallin and NAD~+ isocitrate dehydrogenase alpha(α-NAD~+-IDH) were down-regulated. The result of normal/hypoxia RPE cell showed that there obtained 578 protein spots (matching rat within group was 92.90%) in normal RPE group, and hypoxia group 559 (91.41%). The average matching rate between two groups was 85.47%. There were 92 differential protein spots, which's volume value changed ≥ 2.0 times. 7 spots were selected randomly for MS, and 5 proteins were identified successfully: HSP70 and HSP60 up-regulated; while β -actin, β -tubulin and proxiredoxin 3 down-regulated.[Conclusion] This study was the first research, to our knowledge, which investigates the differential expressional proteins in CNV rats using proteomic technique; and Maps of proteins expression were made in retina-choroid complex in normal/CNV rat and normal/hypoxia human RPE in vitro.There were many proteins changed in the process of CNV in rat; some of those regulated proteins can lead to protein synthesis productively, increasing mitochondrion functions, up the ability of antioxygen, while the aerobic metabolism level in cell down-regulated.Findings of the in vitro study provide the evidence for expression changes of many proteins in RPE cell with hypoxia state; some of those regulated proteins can result in increasing stress capability obviously in cells, while the major cytoskeletal proteins down-regulated, and the ability of sustained-shapes, keeping order internal structure in cells decreasing.This is the first time to find that HSP60 up-regulated in hypoxia state in human RPE cell in vitro, and the advance investigation about this protein maybe discover a new way of CNV genesis.Proteome analysis provides a useful platform in systematic screening of various protein expressions in CNV related diseases.