The effects of mitogen-activated protein kinase on EGF-induced expression of integrin α_5 in human RPE cells / 眼科研究

Background Human retinal pigment epithelial(RPE) cells play a critical role in the pathogenesis of proliferative vitreoretinopathy(PVR) and other related ocular diseases.Research demonstrated that epidermal growth factor(EGF) stimulates activation of RPE cells and therefore causes PVR,and integrin α_5 mediates the adhesion of cells and EGF.Objective This study aims to investigate the role of mitogen-activated protein kinase(MAPK) in regulation of EGF on integrin α_5 expression in human RPE cells.MethodsHuman RPE cells strain(CRL2302) was cultured in DMEM/F12 with 10% calf serum and passaged in 0.25% trypsin.Cultured cells were divided into DMEM/F12control group,20μmol/L PD98059 +10 ng/mL EGF＋DMEM/F12(PD98059) group and 10 ng/mL EGF＋DMEM/F12(EGF) group.The expression of integrin α_5 protein in CRL2302 cells was detected by RT-PCR and calculated as α_5 mRNA/β-actin mRNA,and the expression of integrin α_5 mRNA in CRL2302 cells was detected evaluated by flow cytometry.The MAPK phosphorylation level in each group of human RPE cells was tested by Western blot.ResultsThe expression value of integrin α_5 mRNA was 0.93±0.06 in the control group,1.06±0.07 in PD98059 group and 1.97±0.09 in EGF group,showing a significant difference among the three groups(F=458.896,P<0.01).The fluorescence intensity of integrin α5 protein in CRL2302 cells after 24 hours was 1.94±0.22,4.56±0.25,2.39± 0.14 in three groups respectively with a significant difference(F=21.05,P<0.05).After 30 minutes of culture,Western blot result showed that the strongest phosphorylation levels of ERK1/2 activation in EGF group and the weakest phosphorylation levels of ERK1/2 activation in the control group;While that in PD98059 group was significantly stronger than control group and weaker than EGF group(F=143.14,P<0.01).ConclusionEGF stimulates activation of ERK1/2 pathway in human RPE cells and the expressions of integrin α_5 mRNA and protein in human RPE cells in vitro.

The Effect of Antiproliferative Drugs on the Collagen Matrix Cultured with Retinal Pigment Epithelial Cell and Choroidal Fibroblast

PURPOSE: Epiretinal membrane in proliferative vitreoretinopathy (PVR) may cause tractional retinal detachment after vitreoretinal surgery. It has been thought that the proliferative membrane is mainly composed of choroidal fibroblasts and retinal pigment epithelial cells. Inspite of the technical advances, the treatment of PVR is still difficult. Therefore, the need for phamarcologic treatment of proliferative vitreoretinopathy is increasing. METHODS: In vitro models of proliferative vitreoretinopathy allow to identify the factors which may inhibit proliferation and contraction of collagen matrix by choroidal fibroblast and retinal pigment epithelial cells. Cultured choroidal fibroblasts and the RPE cells were plated to the collagen matrix and antiproliferative drugs was tested. RESULTS: Each antiproliferative drug showed the inhibition of collagen matrix contraction at following concentration: colchicine(0.1 microgram/ml), puromycin(1~10 microgram/ml), cytochalasin B(0.05 microgram/ml). Transmission electron micrograph of collagen matrices showed dense collagen fibers surrounding choroidal fibroblast and fine collagen fibers surrounding RPE cell. Scanning electron micrograph of collagen matrices contaning colchicine, puromycin, or cytochalasin B showed that collagen fibers were well preserved without distortion. All collagen matrices containing RPE cells showed more fine collagen fibers than those containing choroidal fibroblasts. CONCLUSION: Colchicine, puromycin, cytochalasin B showed inhibitory effect on cell mediated contraction in addition to potent antiproliferative effect. Retinal pigment epithelial cell played less significant role in causing PVR than choroidal fibroblast.

Change of Surface Carbohyd rate during Trans differentiation of Retinal Pigment Epithelial Cell

This study was conducted to study the changes of cell surface carbohydrates during transdifferentiation of retinal pigment epithelial(RPE)cells. RPE cells were cultured from adult pig eyes. Surface carbohydrates of RPE cells from 1st, 3rd, 5th, 7th and 9th passages were assayed by lectin histochemistry and enzyme immunoassay. Changes in binding affinities to the lectins employed were demonsrated during trasdifferentiation of RPE cell. Whereas binding affinities of ConA, ECL, PNA, WGA, and UEA-I decreased graudally as the number of culture passage increased, binding properties to LCA, STL and DBA fluctuated depending on the number of passages. The results demonstrate changes of surface carbohydrates of RPE cell during trasdifferentiation. We suggest that changes of surface carbohydrates of RPE cell during trasdifferentiation may be close relations with the functional changes during transdifferentiation.

Effect of Conditioned Medium of Cultured Retinal Pigment Epithelium on Proliferation of Vascular Endothelial Cells in Bovine; In Vitro Study

Neovascularization is a serious complication and major cause of visual loss in ocular diseases such as diabetic retinopathy and proliferative vitreoretionpathy. So there have been many attempts to inhibit neovascularization. Most inhibitors of neovascularzation so far identified have been extracted from lens and vitreous, and recently retinal pigment epitheliumin in culture also secrete angiogenic inhibitors. We cultured retinal pigment epithelial cell, vascular endothelial cell of Umblical vein and subconjunctival fibroblast of bovine. We studied the inhibitory effect in each of retinal pigment epithelium conditioned medium (RPE-CM) and fibroblast-CM on proliferation and migration of vascular endothelial cell comparing the effect of normal media group. In RPE-CM, proliferation and migration of vascular endotrelial cells were inhibited and the inhibition was dose-dependant. But in fibroblast-CM, we didn't observe the inhibitory effect These results suggest that in RPE-CM, there were certam substances to inhibit vascular endothelial cell proliferation and migration. Further research should be continued to detect that substance in molecular biological level.

Effect of Cryotherapy on Proliferative Vitreoretinopathy(PVR)

Cryotherapy is blamed for inducing or aggravating PVR, by releasing retinal pigment epithelial(RPE) cells. These are based on the fact that PVR rarely occurs in non-operated eye, and many of PVR patients have received cryotherapy during surgery. Nontheless, in eyes with diathermy also developed PVR, and although there have been many experiments, the effect of cryotherapy on inducing PVR was not proven experimentally in the living eye. We made retinal tears in the living rabbit eyes, and applied cryotherapy on one eye of each rabbit. The result was compared histopathologically with noncryothermized control eye. There was no statistically significant difference between the two groups concerning the migration of RPE, and the proliferation of RPE. Although the formation of epiretinal membrane was more obvious in the cryothermized group, the difference was not statistically significant.

The effect of cryotherapy on proliferative vitreoretinopathy (PVR)

Cryotherapy is implicated for inducing or aggravating proliferative vitreoretinopathy (PVR) by releasing retinal pigment epithelial (RPE) cells. These are based on the fact that PVR rarely occurs in a non-operated eye, and many of the PVR patients have received cryotherapy during surgery. Nonetheless, eyes with diathermy also developed PVR, and although there have been many experiments, the effect of cryotherapy on inducing PVR has not been proven experimentally in the living eye. We made retinal tears in living rabbit eyes, and applied cryotherapy on one eye of each rabbit. The result was compared histologically with the contralateral noncryothermized control eye. There was no statistically significant difference between the two groups concerning the migration of RPE, and the proliferation of RPE. Although the formation of an epiretinal membrane was more obvious in the cryothermized group, the difference was not statistically significant.