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 divid ed cells as the control were dissociated with routine normal culture medium cent rifugation. The Trypan blue (0.4%) rejection staining was used, and the mouse anti-human monoclonal anti-body 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＜0.001）, but the number of the cells was reduced. The cells in the experimental group were integrated round with smooth border, symmetrical staining, homogeneous configuration and higher rate of clone formation （P＜0.001）. Conclusions RPE cells disas sociated with ficoll gradient centrifugation have the better dissociation effects. (Chin J Ocul Fundus Dis，2003，19：333-404)
Purpose To investigate bax expression and induction of apoptosis in normal cultured retinal pigment epithelium (RPE) cells ． Methods Cultured human RPE cells were transfected by PMDNA3-hbax，which incoded the whole bax gene and may be induced by Zn2+ under the MTII promoter， through lepofectin mediated protocol．The tested RPE cells were divided into three groups of A，PMDNA3-hbax transfected ;B，PMDNA3 (nude vector) transfected and C ，normal RPE cells．After transfection， DNA gel electrophoreses were perform ed ，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 PMDNA3-hbaxtransfected ， 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 in to the RPE cell through lepofectinmediated protocol and induced expression ． The foreign bax overexpression may induce the cultured human RPE cell susceptibi lity to apoptosis． (Chin J Ocul Fundus Dis, 2001,17:132-134)
Purpose To study inhibition effects of retinal pigment epithelial (RPE) cells by hyaluronic acid-stimulating activity(HASA)． Methods The cultured human RPE cells added with a series of HASA was measured with cell counting,tetrazolium(MTT)colorimetric assay and tritium labelled thymidine deoxyribosｅ(3H-TdR)incorporation assay.Flow cytometry(FCM)analysis was used to examine RPE cells cycles. Results HASA at concentrations of 12.5～200 mu;g/ml and within 48 hours inhibited RPE cells proliferation with a dose-dependant and time dependant manners.The maximal inhibition rate of RPE cells by HASF was about 48.0%.FCM revealed that the cells in G1 phase increased 7.2% and cells in S phase decreased 9.7%,compared to controls. Conclusion HASA at a certain dose range and period can inhibit RPE cells proliferation. (Chin J Ocul Fundus Dis,1999,15:72-74)