| Purpose Proliferative vitreoretinopathy (PVR) is anexcessive wound healing response which is a very complex cytoreticulum procedure involving many kinds of cells and cytokines. But several vital cells or cytokines may play an important role in this procedure. Migration and proliferation of retinal pigment epithelial (RPE) cells are key steps in the pathological process in the development of PVR. Epidermal growth factor receptor (EGFR) is a cell surface receptor with intrinsic tyrosine kinase activity. The engagement of the receptor by its ligand can induce intracellular mitogenic signal transduction pathways and stimulate cells proliferation, migration, differentiation, et al. Therefore, studying the role of EGFR in PVR may be give a new treatment strategy for PVR.This experiment aimed to: (1)To investigate the expression of EGFR in periretinal membranes of PVR and the origination of EGFR-positive cells; (2)To examine the effects of epidermal growth factor (EGF) and fetal calf serum (FCS) on the proliferation andmigration of cultured human RPE cells; (3)To detect the activation and role of EGF-EGFR-MAPK signal transduction pathway in proliferation of human RPE cells. Methods(1) 43 periretinal membranes of PVR obtained during vitrectomy were divided into 3 groups according the time course of PVR: the early-stage membranes (<2 months), the middle-stage membranes(2~6 months) and the late-stage membranes(>6 months), which were studied by immunohistochemical staining and in situ hybridization(ISH) in order to investigate the expression of EGFR. Furthermore, the original of EGFR-positive cells were detected with immunofluorescence histochemical double-staining technique.(2) Cultured human RPE cells of 3th~6th passages were studied by colorimetric assay for cellular growth and survival (MTT assay) to investigate the effects of EGF (0.1 ng/ml 1 ng4nl 10ng/ml 50ng/ml 100ng/mll) and FCS on proliferation of human RPE cells; We used an in vitro wound healing model and counted the number of cells that had entered the denuded area to assess the effects of EGF and FCS on the migration of human RPE cells.(3) The cultured human RPE cells had been stimulated with 0.1%FCS, 10%FCS, 10ng/ml EGF+0.1%FCS and with a combination of EGF and 10%FCS for 3 days. Immunohistochemical staining and in situ hybridization were used to observed the expressions of EGFR protein and EGFR mRNA, respectively. Activation of MAPK was detected by immunohistochemical method with specific anti-phosphorylated ERK1/2 antibody.Results (1) Immunohistochemical study showed that EGFR had strong positive staining in the early-stage membranes, weak staining in the middle-stage membranes and negative expression in the late-stage membranes. The results of ISH proved that this receptor gene only expressed positive staining in the early-stage membranes of PVR. The EGFR-positive cells showed positive staining to cytokeratinpan and macrophages marker HAM-56, but negative staining to glial fibrillary acidic protein (GFAP).(2) EGF stimulated proliferation and migration of cultured human RPE cells in a concentration-dependent manner. The maximal proliferation rate of RPE cells was 81.8% at 10ng/ml -100ng/ml EGF in DMEM and 122.7% at 1ng/ml-10ng/ml EGF in 5%FCS DMEM; There is significantly difference in DMEM groups and 5% FCS DMEM groups (P<0.001), moreover, 5%FCS notably induced human RPE cells proliferation (P<0.001). 1ng/ml-100ng/ml EGF slightly stimulated human RPE cells migration; The maximal migration rate of RPE cells was 438.9% at 10ng/ml -100ng/ml EGF in 10%FCS DMEM; 10% FCS induced human RPE cells migration( the migration rate, 147%) stronger than lng/ml-100ng/ml EGF did alone(the maximal migration rate, 36%); the migration rate of 10% FCS DMEM groups has significantly higher than that of DMEM groups(P<0.001).(3) EGF promoted the expression levels of EGFR protein and EGFR mRNA of RPE cells. FCS not only could cooperate with EGF, but also could induce the expression of EGF...
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