[Screening and identification of abnormal expression proteins from hypoxia human retinal pigment epithelium cell in vitro].
OBJECTIVE
To screen and identify the abnormal proteins expressed by hypoxia human retinal pigment epithelium (RPE) in vitro.


METHODS
It was a experimental study. Protein of normal/hypoxia human RPE cells in exponential phase of growth was extracted, and frozen by liquid nitrogen for proteome study. All samples were 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/hypoxia RPE cell. For identification of differential proteins, peptide masses were analyzed by matrix-assisted laser desorption ionization time-of flight mass spectrometry (MALDI-TOF/MS).


RESULTS
Five hundred and seventy-eight protein spots were obtained in normal group (matching rate within group was 92.90%), and hypoxia group 559 (91.41%). The average matching rate between two groups was 85.47%. There were 32 differential protein spots, which volume value changed > or = 2.0 times. 7 spots were selected randomly for MS, and 5 proteins were identified successfully: HSP70 and HSP60 up-regulated; while beta-actin, beta-tubulin and peroxiredoxin 3 down-regulated.


CONCLUSIONS
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. Proteome analysis provides a useful platform in systematic screening of various protein expressions in CNV related diseases.
He
Shou-zhi
SZ
Department of Ophthalmology, General Hospital of PIA, Beijing 100853, China. heshouzhi@sina.com
Hou
Bao-ke
BK
Tang
Wei-qiang
WQ
Ding
Zhen-qiang
ZQ
D015687:Cell Hypoxia; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000378:metabolism; D020256:Choroidal Neovascularization / Q000378:metabolism* / Q000473:pathology; D015180:Electrophoresis, Gel, Two-Dimensional; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D020543:Proteome; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018834:Chaperonin 60; D050883:HSC70 Heat-Shock Proteins; D020543:Proteome