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Proteomic analysis of soluble factors secreted by limbalfibroblasts
[摘要] Purpose: To identify soluble factors selectively secreted by limbalfibroblasts as possible regulators of limbal basal epithelium.Methods: Limbal, corneal, and conjunctival fibroblasts were firstexpanded in vitro in Dulbecco's modified Eagle medium containing 10%fetal bovine serum, and then maintained in serum-free medium for twoweeks. Proteomic analysis of culture supernatants was done to comparedifferences in secreted matricellular proteins. Real time PCR andwestern blots were done to confirm the expression of secreted proteinacid and rich in cysteine (SPARC), a protein found in abundance inextracellular proteins secreted by limbal fibroblasts.Immunohistochemistry of SPARC was done in human limbal tissue to showthe spatial distribution of the protein. An adhesion assay was designedto demonstrate the effects of SPARC on an SV40 immortalized humancorneal epithelial cell line (HCEC).Results: Proteomic analysis revealed several proteins selectivelysecreted by limbal fibroblasts. The particular spots were identified asSPARC, vimentin, serine protease, collagen alpha 2 precursor, tissueinhibitor of metalloproteinase 2 (TIMP-2), and5,10-methlenetetrahdrofolate reductase (FADH2). The expression of SPARCwas confirmed by western blot analysis, and mRNA expression wassignificantly higher in limbal fibroblasts compared to central cornealfibroblasts when analyzed by real time PCR. Immunohistochemistryrevealed higher distribution of SPARC in the subepithelial stroma of thelimbus compared to the central cornea. The addition of 10 μg/mlmurine SPARC in HCEC significantly reduced cell spreading at three h.Conclusions: The matricellular protein SPARC is preferentiallysecreted by limbal fibroblasts, and may modulate intercellular adhesionof basal limbal epithelial cells.
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[效力级别]  [学科分类] 生物化学/生物物理
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