| Background and Purposes Connective tissue growth factor (CTGF) was originally identified in conditioned medium from human umbilical vein endothelial cells. It is a 38-KDa protein and a secreted cysteine-rich heparin-binding polypeptide growth factor, belong to one of the members of the CCN family of immediate early genes. Now it has been shown to be widely existed in many human tissues and organs and synthesized by a variety of cells including fibroblasts, some vascular endothelial and vascular smooth muscle cells, epithelial cells, tumor cells, and chondrocytes. Biological effects of CTGF include stimulation of mitosis and proliferation, cell migration, cell attachment, angiogenesis and extracellular matrix (ECM) production. CTGF has recently received much attention as a possible key determinant of progressive fibrosis and excessive scarring and also of wound repair, neoangiogenesis, tumor growth and embroyonic development. The expression of CTGF was shown to be selectively stimulated by transforming growth factor-P (TGF-P),and partly acts as a downstream mediator of TGFP-induced biological functions.Proliferative vitreoretinopathy (PVR) is a common sequel to rhegmatogenous retinal detachment (RRD) and is the leading cause of failed surgery repair. It is well known that retinal pigmentepithelial (RPE) cells are predominant proliferative cells in PVR which is an excessively wound healing response occurring after RPE cell wounding. The activated RPE cells migrate from their normal, sessile state to vitreous and onto both surfaces of the retina, in which they begin to dedifferentiate, migrate, proliferate, change phenotype and secrete ECM. Multiple proteins and growth factors are involved in this process. But it is not completely clear that the mechanism of modulation on RPE cells growth in the pathogenesis of PVR, which delays the development of prevention and treatment of PVR.The widely biological behavior of CTGF is in concordance with the change of RPE cells in PVR, and CTGF protein and mRNA had been shown to exist in PVR proliferative membranes, but it has not been reported about the expression and function of CTGF in vitro cultured human RPE cells at present. The aim of our experiments is to investigate the relationship between CTGF and transforming growth factor-P receptor II (TGF- P RII) and ECM, the expression and modulation of CTGF in cultured human RPE cells, the effects of CTGF on migration, adhesion, proliferation and ECM synthesis of RPE cells, and observe the effect of CTGF on calcium signal transduction pathway. Methods1. Immunohistochemistry was used to detect the expression of CTGF, TGF-PRII, FN, Collagen I and collagen III protein, in site hybridization(ISH) was used to detect the expression CTGF mRNA and TGF-pRIImRNA in periretinal membranes(PRM)of PVR, and immunofluorescence histochemcial double-staining technique was used to identify the origination of CTGF-positive cells in PRM. The correlations of the expression of CTGF, TGF-PRIIand ECM were analyzed by statistics.2. (1) Immunofluorescence cell staining, western blot and RT-PCR technique were used to investigate the expression of CTGF protein and mRNA and its modulation by different growth factors stimulation in cultured human RPE cells. (2) A vitro model of RPE monolayer wound healing which mimics the proliferative and migratory responses of RPE cells in PVR was used to examine the expression of CTGF protein and mRNA by western blot and ISH.3. CD The model of RPE wound healing was used to examine the effects of recombinant human CTGF (rhCTGF) or combination with dexamethasone (DEX) on modulation of the migration of RPE cells. (2) A 96-well microtiter plates were coated with CTGF, collagen protein and Poly-L-lysine(PLL) respectively to observe the effect of CTGF on cell adhesion. (3) Proliferation in RPE cells was evaluated using MTT assay and flow cytometry (FCM). (4) To determine the cytoplasmic free Ca2+ concentration ([Ca2+] i) response to CTGF, the fluo-3/AM loaded RPE cells were observed using a laser scanning confocal... |
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