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Metforminin invitro retina pigment epiteli hücrelerinde fibrogenezise olan etkisinin incelenmesi / Investigation of the effect of metformin on fibrogenesis in invitro retina pi̇gment epi̇theli̇al cells

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Date
2019
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ÇETİNKAYA, Rukiye
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Bezmialem Vakıf University
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Abstract
Proliferative vitreoretinopathy is one of the most important causes of recurrence after retinal detachment surgery. In studies on the pathogenesis of proliferative vitreoretinopathy, ''Epithelial-to-Mesenchymal Transition'' has been shown to play an important role. Epithelial-to-mesenchymal transition; it is a biological process in which epithelial cells lose their differentiated phenotypes and become mesenchymal-like cells. Retinal pigment epithelial cells undergoing epithelial-to-mesenchymal transition; proliferation, migration and contraction ability increases. Fibrosis, an important part of proliferative vitreoretinopathy, develops at the end of this process. The main trigger of epithelial-to-mesenchymal transition is TGF-β. PURPOSE: In our study, the effect of metformin which has been shown to inhibit epithelial-to-mesenchymal transition and fibrogenesis in different tissues previously, on TGF-β2-induced epithelial-to-mesenchymal transition process in invitro retinal pigment epithelial cells was investigated. MATERİALS abd METHODS: TGF-β2, metformin and TGF-β2 + metformin were applied to human retinal pigment epithelial cell groups incubated in vitro. After the application; cell viability by ATP test, ZO-1, α-SMA, vimentin, SMAD2, SMAD3, pSMAD2 and pSMAD3 protein expression by Western Blot Protein Electrophoresis, cell migration level by wound healing test and contraction level by collagen gel contraction test were evaluated, the data were compared with the control group. RESULTS: In Western Blot protein electrophoresis, the expression of ZO-1, which is an epithelial marker, decreased significantly in the TGF-β2 group compared to the control group (p <0.05), but there was no statistically significant difference in the TGF-β2 + metformin group compared to the control group. It was observed that α-SMA expression, which is a mesenchymal marker, was significantly increased in the TGF-β2 group compared to the control group (p <0.001), whereas this increase was lower in the TGF-β2 + metformin group (p <0.01). The expression of vimentin, which is a mesenchymal marker, was significantly higher in the TGF-β2 group than in the control group (p <0.001), but this increase was less in the TGF-β2 + metformin group (p <0.05). When the expression of pSMAD2 and pSMAD3, the active proteins of the TGF-β / SMAD signaling pathway were evaluated, it was found that pSMAD2 increased significantly in the TGF-β2 group compared to the control group (p <0.001) and decreased significantly in the TGF-β2 + metformin group compared to the control group. (p <0.05). It was found that pSMAD3 increased significantly in TGF-β2 group compared to the control group (p <0.001), and there was no significant difference in TGF-β2 + metformin group compared to control group. In wound healing test results; There was a significant increase in cell migration level in TGF-β2 group compared to the control group (p <0.001). There was no difference in TGF-β2 + metformin group compared to the control group. Collagen gel contraction test results; The level of contraction was significantly higher in the TGF-β2 group than in the control group (p <0.001), whereas this increase was less in the TGF-β2 + metformin group (p <0.05). CONCLUSION: Metformin increased the synthesis of epithelial markers and decreased the synthesis of mesenchymal markers in retinal pigment epithelial cells undergoing epithelial-to-mesenchymal transition. Metformin suppressed the TGF-β / SMAD signaling pathway by reducing phosphorylation of SMAD2 and SMAD3 proteins. In addition, it has been observed that metformin reduces cellular migration and contraction levels of retinal pigment epithelial cells undergoing epithelial-to-mesenchymal transition. These results show that metformin suppresses epithelial-to-mesenchymal transition and development of fibrogenesis in invitro retinal pigment epithelial cells. KEYWORDS: Metformin, Proliferative Vitreoretinopathy, Epithelial-to-Mesenchymal Transition, Retinal Pigment Epithelial cells, TGF-β2
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Thesis (Medical)--Bezmialem Vakıf University, Faculty of Medicine, Department of Eye Diseases, Istanbul, 2019
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Göz Hastalıkları = Eye Diseases
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