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M. Shibuya, Y. Tanaka, J. Utsumi, M. Mutoh, V.N. Reddy, T. Iwata; Characterizationof Novel Growth Promotive Factor for Retinal Pigment Epithelial Cells, REF–1/TFPI–2 . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1298.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have previously isolated a growth promotive factor REF–1/TFPI–2, which specifically stimulated the proliferation of human retinal pigment epithelial (RPE) cells. To observe the internal and external effect on human RPE cells by REF–1, two experiments were performed. ELISA assay of cytokines secreted from human RPE cells after REF–1 treatment versus non–treatment and DNA microarray analysis of total RNA isolated from REF–1 treated versus non–treated human RPE cells were performed. Methods: The relationship between RPE cell growth and cytokines production of bFGF, transforming growth factor–ß1 (TGF–ß1), transforming growth factor–ß2 (TGF–ß2), interleukin–1α (IL–1α), interleukin–6 (IL–6), interleukin–8 (IL–8), tumor necrosis factor–α (TNF–α), epidermal growth factor (EGF), granulocyte–colony stimulating factor (G–CSF), granulocyte–macrophage CSF (GM–CSF), and macrophage–CSF (M–CSF) by REF–1 treated and non–treated human primary RPE cells were examined. The cells were grown in DMEM medium with 15% FCS for 3 days and then replaced by serum–free DMEM medium. After 2 days, medium was replace again by REF–1 containing (10ng/ml) serum free DMEM. After 3 additional days, cytokines in the culture supernatant was determined by an ELISA kit (Amersham Biosciences, R&D Systems, Imuuno–Biological Laboratories). Under the same experimental condition total RNA was isolated after 6, 12, 24h of REF–1 treated and non–treated human primary RPE cells. DNA microarray analysis was performed using Applied Biosystems 1700 Chemiluminescent Microarray Analyzer. Genes affected by REF–1 treatment was characterized using Celera Discovery System (Applied Biosystems). Results:Among the measured cytokines, TGF–beta1, GM–CSF, IL–8, M–CSF, and IL–6 were increase by 4.7, 2.4, 1.5, 1.2, and 1.1–fold, respectively. DNA microarray analysis also confirmed transcriptional up–regulation of these cytokines. Conclusions:ELISA assay and DNA microarray analysis confirmed various cytokines up–regulated and secreted by REF–1 treatment. Among these cytokines TGF–ß1 and GM–CSF were significantly induced. The mechanism of how these cytokines relates to the specific stimulation of the proliferation of RPE cells is still unknown. However this property make it potentially beneficial for the intraocular therapy for the repair and maintenance of RPE cells in various retinal diseases.
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