| Objective To investigate the significance and regulation of epidermal growth factor (EGF) on human retinal pigment epithelial cells'(hRPE) integrinα5 expression.Method 1) We collected 14 subretinal membrane specimens by vitrectomy of retinal detachment cases.And detected the expressions of keratin, integrinα5β1 and fibronectin (FN) by immunohistochemical methods. We studied the relation between retinal pigment epithelial cell membranes and expression of integrinα5β1 by double-labeling immunofluorescence,and analyzed the difference of integrinα5β1 expression of retinal membrane with the membrane compared with normal by fluorescence quantitative. 2)We used human retinal pigment epithelial cells of primary and mass culture.The hRPE cells were induced by EGF of different concentrations (0ng/ml,0.1ng/ml, 1ng/ml, 10ng/ml, 20ng/ml,100ng/ml).We observed the integrinα5 mRNA and protein expression by RT - PCR, immunohistochemistry and flow cytometry .We measured cells proliferation rates of the experimental groups by MTT assay,and tested cells migration with a diameter of 8.5mm Boyden chamber. 3) There were three groups: the control group, the 10ng/mlEGF group and the PD98059 group. We observed the integrinα5 mRNA and protein expression of different groups by RT-PCR and flow cytometry, and tested hRPE cells MAPK phosphorylation level in each group by Western blot.Result 1) All 14 cases were positive expression of integrinα5β1 and fibronectin (FN) protein,the RPE cells with keratin expressedα5β1 on the subretinal membrane, compared with the normal the expression significantly enhanced(P <0.01). 2)Compared with 0ng/ml group, the 1ng/ml of EGF promoted integrinα5 mRNA and protein expression in hRPE cells after 24 hours and with a concentration-dependent manner;and the role peaked at concentrations of 10 to 100ng/ml. Flow cytometry showed that the 10ng/ml of EGF played the strongest role in integrinα5 induction;and the fluorescence intensity was 3.98±0.67.When the concentrations were 0ng/ml and 0.1ng/ml,they were1.87±0.22, 1.98±0.54 respectively (P <0.01). MTT assay showed results as follows: compared with the control group,cells proliferated and migrated obviously in EGF-induced group and not-related antibody (rabbit anti-human antibody vimentin) one;the role was weak in rabbit anti-human integrinα5 polyclonal antibody (1:100) group; and the difference was significant (P <0.01). 3) Compared with the control group, EGF could promote expression of integrinα5 mRNA and protein, but PD98059 (20μmol/L) inhibited this role. Flow cytometry showed the integrinα5 fluorescence intensity after 24 hours were 1.94±0.22, 4.56±0.25, 2.39±0.14, and the difference was significant (P <0.05). After 30 minutes: Western blot results showed that the highest phosphate levels of ERK1/2activation in EGF-induced group, and the control group of the ERK1/2 phosphorylation activated weak, but the PD98059 inhibited the activation.Conclusion The hRPE cells in the subretinal membranes up-regulated expression of integrinα5β1, and its ligand FN also notably expressed ; and so the integrinα5β1 and FN played the role in the retinal membrane formation. 1ng/ml of EGF promoted integrationα5 mRNA and protein expression and the promotion reached a peak of 10~100 ng/ml; and the expression of integrinα5 in hRPE promoted cells proliferation and migration. ERK1/2 phosphorylation participated in the activation process. EGF stimulated activation of ERK1/2 pathway in hRPE cells, and it promted the integrinα5 mRNA expression in playing an important role in PVR.
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