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Objective To investigate the effect of dissociation of the human retinal pigment epithelium (RPE) cells by ficoll hypaque gradient centrifugation. Methods The primary human RPE cells and subcultured human RPE cells were dissociated with ficoll gradient centrifugation solution (d= 1.077 g/ml) and the same divided cells as the control were dissociated with routine normal culture medium centrifugation. The Trypan blue (0.4%) rejection staining was used, and the mouse anti-human monoclonal antibody and fluorescein isothiocyanate (FITC) labeled rabbit anti-mouse IgG were utilized for indirect immunoreactivity for the test of human cytokeratin (CK) in active RPE cells cytoplasm. Flow cytometry assay was used to analyzed the percentages of CK positive staining RPE cells. simultaneously, the cells configuration, growth condition, the rate of clone formation, and the purifying result were observed under the fluorescent and confocal microscope. Results The survival rate and positive rate of CK of RPE cells in experimental group were higher than those in the control ( P
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Objective To investigate the activation and role of signal transduction pathway of epidermal growth factor (EGF)-epidermal growth factor receptor (EGFR)-mitogen activated protein kinase (MAPK) in proliferation of human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were stimulated with 0.1%,10% foetal calfserum (FCS) and EGF(0.1, 1, 10, 50 and 100 ng/ml)in 0.1% FCS Dulbeco′s modified Eagle′s medium (DMEM) and in 10% FCS DMEM for 3 days, respectively. Immunohistochemical staining and in situ hybridization were used to observe the expressions of EGFR protein and EGFR mRNA,respectively. Activation of MAPK was detected by immunohistochemical method with specific anti-phosphorylated ERK 1/2 antibody. Results The optimal concentrations of EGF were 10 ng/ml in 0.1% FCS DMEM and 1 ng/ml in 10% FCS DMEM. After 3 days of stimulation with EGF, phosphorylated ERK 1/2 staining was detectable in nucleus of RPE cells, whereas cells presented immunostaining for phosphorylated ERK 1/2 in the cytoplasm before stimulation. Conclusions EGF may improve the expression of EGFR protein and EGFR mRNA of RPE cells, and induced MAPK nuclear translocation in a concentration-dependent manner. EGF-EGFR-MAPK signal transduction pathway may play a key role in RPE cells proliferation, and serum exerts an important acceclerating function in the process.
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Purpose To investigate bax expression and induction of apoptosis in normal cultured retinal pigment epithelium (RPE) cells . [WT5”HZ]Methods [WT5”BZ]Cultured human RPE cells were transfected by P MDNA3 hbax ,which incoded the whole bax gene and may be induced by Zn 2+ under the MTII promoter, through lepofectin mediated protocol.The tested RPE cells were divided into three groups of A, P MDNA3 hbax transfected ;B,P MDNA3 (nude vector) transfected and C,normal RPE cells.After transfection, DNA gel electrophoreses were performed ,the tested RPE cell cycles were analyzed with flow cytometry (FCM). Results The gel electrophoretogram showed DNA ladder phenomenon,FCM confirmed the apoptosis of RPE cells P MDNA3 bax transfected , consisting of significant apoptotic peak sited before the G 1 phase and the apoptotic rate was 36%. Conclusion The foreign bax gene can be effectively conducted into the RPE cell through lepofectin mediated protocol and induced expression . The foreign bax overexpression may induce the cultured human RPE cell susceptibility to apoptosis.