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Proteomics Study in Human Vitreous Humor of Proliferative Retinopathy

Author: ZhangZuo
Tutor: ZhangXueDong
School: Chongqing Medical University
Course: Ophthalmology
Keywords: Proliferative vitreoretinopathy Vitreous body Two-dimensional gel electrophoresis Mass Spectrometry Proteome
CLC: R776.4
Type: Master's thesis
Year: 2008
Downloads: 12
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Abstract


Objective: (1) to establish and optimize the vitreous (vitreous humor, VH) Two-dimensional gel electrophoresis - mass spectrometry. (2) the use of two-dimensional gel electrophoresis (2-DE) proliferative vitreoretinopathy (proliferative vitreoretinopathy, PVR) eye with healthy donated eye (control group), VH protein expression profiling to compare to find PVR VH differences in protein. (3) The combination of peptide mass fingerprinting technology (PMFs) differential protein spots were identified and data search. Clarify the differences in protein and PVR relationship, looking for disease-specific protein PVR (DSPs), to provide a new method for the diagnosis and drug treatment of PVR. Methods: (1) by vitrectomy surgery collected undiluted the PVR patients with VH for the experimental group, eyes VH healthy donors as a control group. (2) to extract the total protein in the experimental group and the control group VH. (3), respectively, with cold acetone precipitation, TCA / acetone precipitation purification VH protein concentrate immobilized pH gradient - isoelectric focusing (IPG-IEF) to the vertical by SDS-PAGE electrophoresis second to two-way condensate two-dimensional gel electrophoresis (2-DE) VH protein separation, and specimen processing ,2-DE optimization. (4) collection of the gel images, PDQuest software for image analysis, to find meaningful protein spots. (5) remove the differential protein spots, enzymatic hydrolysis, mass spectrometry (MS) identification, peptide mass fingerprint the spectrum (PMF), Mascot and MS-fit software for Internet data retrieval, to find differences in protein. Results: (1) comparing two sample purification method for concentrating found that initial extraction of the VH protein of cold acetone precipitate obtained satisfactory electrophoresis sample concentration and purity. (2) compare the different electrophoretic separation effect of the pH range, the VH protein is concentrated in the range pH4-7, using pH7-4 of the IPG strips is always better separation. (3) compare the sample volume obtained 2-DE gels, that the the sample volume 300μg Finally the silver staining 17cmIPG strip is the most appropriate to obtain a high-resolution 2-DE gels. (4) After optimization of electrophoretic conditions to obtain reproducible 2-DE gels, 12 protein spots on 2-DE maps of experimental and control groups were analyzed. The experimental group, nine high expression and low expression of three. (5) on the differentially expressed proteins identified by mass spectrometry and identified four possible different proteins associated with PVR, respectively, as the guanine nucleotide binding protein (Guanine nucleotide-binding protein G, G protein), sorting Connexin 11 (Sorting nexin 11, SNX11), the class optical sensing factor protein (Phosducin-like protein, PHLP), S100-A7 protein. G protein S100A7 upregulated in the experimental group, down of SNX11 PHLP expression in the experimental group. Conclusions: (1) In this study established a stable clinical the vitreous specimen collection method, successfully established and optimized the VH protein 2-DE/MS technology. (2) The experimental and control groups of VH protein expression have significant differences, the high expression or low expression of the four proteins in the PVR vitreous, respectively, for the G protein S100A7 protein sorting Connexin 11, the class optical sensing factor protein. (3) obtained in four proteins in function with cell proliferation and differentiation of closely related process variety of PVR cells, especially the abnormal proliferation and differentiation of RPE cells play a role, presumably formed in the PVR.

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CLC: > Medicine, health > Ophthalmology > Lens and vitreous diseases > Vitreous diseases
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