Objective To investigate the effects of transformin growth factor-beta (TGF-beta;) and interferon-gamma(IFN-gamma;)on collagen synthesis in human retinal pigment epithelial cells(RPE)． Methods TGF-beta;(0.01～10 ng/ml),recombinant IFN-gamma;(100～10000 Ｕ/ml)or a combination of two were added to cultures of RPE and collagen synthesis of the cells were measured by3 H-proline incorporation assay,indirect immunofluorescence staining and dot-blot hybridization. Results TGF-beta; at 10 ng/ml increased cell uptake of 3 H-proline to 130．87% of controls．It intensified Type IV,I and Ⅲ collagen fluorescent staining as well as mRNA expression．IFN-gamma; at 10000 Ｕ/ml caused 54．72% inhibition of 3 H-proline uptake by RPE,and decreased TypeⅣ collagen fluorescent staining as well as mRNA expression of Type Ⅳ,I and Ⅲ collagens. Conclusion TGF-beta; and IFN-gamma; stimulated and inhibited collagen synthesis of human RPE,respectively．The combination of two had antagonistic effects．IFN-gamma; can be used for inhibition of collagen synthesis of RPE. (Chin J Ocul Fundus Dis, 1999, 15: 245-248)
ObjectiveTo observe the expression of connective tissue growth factor (CTGF) in injured model of retinal pigment epithelial (RPE) cells and the promoting effect of CTGF on migration of RPE cells.MethodsCultured monolayer-confluent human RPE cells were scraped with a trephine and a cotton stick, and set up the injured model of RPE cells with round scraped area. Immunohistochemistry and in situ hybridization(ISH) were used to detect the expression of CTGF protein and mRNA in injured RPE cells at distinct time points after injury. The number of RPE cells migrated to injured area was measured and the effect of CTGF on migration of RPE cells and the effect of dexamethasone (DEX) on the promoting process of CTGF were observed.ResultsThe results of immunohstochemistry and ISH indicated the weak positive expression of CTGF in RPE cells at the edge of scrape 6 hours after injury, and the positive expression increased gradually as time goes by after the injury. Strong positive expression of CTGF in RPE cells at the edge of scrape was found 24 and 48 hours after injury. Rebuilt human CTGF stimulated migration of RPE cells in a dose-depended manner, and DEX significantly inhabited the migration.ConclusionCTGF involves in the procedure of repair of injury of RPE cells, which may play an important role in the pathogenesis of intraocular proliferative diseases such as proliferative vitreoretinaopathy.(Chin J Ocul Fundus Dis, 2005,21:306-309)
Objective To examine the expression of proliferating cell nuclear antigen (PCNA) of retinal pigment epithelial (RPE) cells, thus assessing the role of mechanism of contact inhibition playing in the process of experimental retinal detachment and reattachemnt.Methods Retinal detachment was produced in 72 cats by subretinal injection of 0.25% solution of healon through a micropipette three weeks after extracapsular lens extraction and vitrectomy. Some of the detached retinae were reattached 24 hours later. At different time, the cats were killed and eye globes were fixed and embeded in paraffin. Histologic sections were processed for immunohistochemistry examination using an antibody to detect PCNA protein. Labeled RPE cells were identified, and the proliferation was quantified in detached and un-detached retinae of detachment group, and also in reattached retinae of reattachment group. The comparsion of PCNA-labeled RPE cells in different groups were analyzed by ANOVA. Results In detached regions of detachment group, PCNA-expression of RPE cells occured within 24 hours, and reached a maximum after 5-6 days, then gradually declined to barely detectable levels after 20 days. Similar tendency was found in reattached retinae, but the number of PCNA-labeled RPE cells was obviously small. Fewer PCNA-labeled RPE cells were found in regions of un-detached retinae in detachment group. The difference of these three groups was significant.Conclusion Proliferation of RPE cells is induced when they lose contact with neural retina, but inhibited after neural retina reattached to RPE cells. It suggests that the mechanism of contact inhibition plays a role in the proliferative process after retinal detachment and reattachment. (Chin J Ocul Fundus Dis，2003，19：20-23)
Objective To investigate the features of ocular fundus of retinal pigment epithelial detachment (PED) in Chinese patients more than 50. Methods The clinical data of 31 continuous patients (34 eyes) with PED diagnosed by ocular fundus photochromy, fundus fluorescein angiography (FFA) and indocyanine green angiography ( ICGA ) from Oct, 2001 to Aug, 2004 were analyzed retrospectively. Results In 34 eyes with PED, the results of FFA showed serous PED in 18 (52.9%), hemorrhagic PED in 8 (23.5%), and serosanguineous PED in 8 (23.5%); the results of ICGA revealed PED associated with choroidal neovascularization (CNV) in 12 (35.3%), PED associated with ploypoidal choroidal vasculopathy (PCV) in 17 (50.0%), PED associated with both CNV and PCV in 1 (2.9%), and avascular PED in 4 (11.8%). Conclusions PED in Chinese patients more than 50 can be associated with CNV, PCV or other avascular diseases, and PCV is the most common intercurrent choroidal vascular disease. (Chin J Ocul Fundus Dis, 2006, 22: 224-227)
Objective To evaluate the toxic effects of staphylococcus aureus exotoxins and neutrophils on retinal pigment epithelium (RPE) cells (RPEC). Methods An in-vitro model of bacteroidal endophthalmitis was established by co-culturing of human RPE cell line D407 and human peripheral blood neutrophils in the present of staphylococcus aureus exotoxins ATCC29213. The level of lactate dehydrogenase hydroxide（LDH）in the cuture supernant was measured, and the viability of RPE was evlauated by flow cytometry and Hoechst 33342/Propidium Iodide（PI）staining. Results When RPE cells were cultured with the exotoxin ATCC29213, the LDH level and necrotic RPE cells were positive proportional to the dosage of exotoxin, but only 250mu;l or 500mu;l of ATCC29213 had a statistical significant effect. When RPE cells were co-cultured with neutrophils in the present of ATCC29213 for 6 hours, 100mu;l of ATCC29213 already had a statistical significant effect on LDH level and necrotic RPEC, and the effect was proportional to the amount of neutrophils in the culture. Conclusion Both staphylococcus aureus exotoxins and neutrophils can damage the RPEC by inducing necrosis, and their function had synergetic effect.
Objective To investigate effects of neural retina on development of the structure of outer blood retinal barrier in embryogenesis. Methods The retinal neural epithelium (RNE) and pigment epithelium (RPE) layers of 150, 120 and 90 embryonic chicken eyes incubated for 7,10, and 14 days were peeled off. RNE was used to prepare the culture medium with different conditions (7drcSF3, 10drcSF3, 14drcSF3). RPE cells of 7- and 14-incubated chicken embryos were cultured on laminin-coated transwell filter. The SF3, 7drcSF3, 10drcSF3 , 14drcSF3 medium were used respectively in the apical chamber and SF2 was used in basolateral chamber. After the formation of monolayer, the transepithelial electrical resistance of the RPE was detected. After the fixation of RPE cells, the condition of the tight junction among the cells was observed by immunohis tochemistry and transmission electron microscopy. Results For the RPE cells of 7-and 14-day incubated embryonic eyes, the difference of TER in various medium of SF3/SF2, 7drcSF3/SF2, 10drcSF3/SF2, 14drcSF3/SF2 was statistically significant (P＜0.01). The polarity of RPE cells was induced and the netlike tight junctional strands was urged in the retina-conditioned medium. Conclusion The neural retina may actively promote the formation of the structure of outer blood retinal barrier. (Chin J Ocul Fundus Dis，2004，20：237-240)
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)
Objective To investigate the expression of hepatocyte growth factor receptor (HGFR) in epiretinal membranes (ERM) of eyes with proliferative vitreoretinopathy (PVR) and cultured retinal pigent epithelium (RPE) cells. Methods Fifteen human epiretinal membranes were obtained from eyes undergone vitrectomy for rhegmatogenous retinal detachment complicated with PVR and observed by immunohistochemical examination to study the expression of HGFR. Using the immunohistochemical technique to evaluate the expression of HGFR in cultured RPE cells. Results In 6 membranes of PVR-grade C, HGFR were expressed in 5/6, and 7 cases were detected in 9 membranes of PVR-grade D.RPE cells express readily detectable levels of HGFR. Conclusion The findings indicate that HGF might be involved in the formation of epiretinal membranes in PVR. (Chin J Ocul Fundus Dis, 2002, 18: 221-223)
Purpose To investigate the effects of human vitreous fluid on proliferation of cultured human retinal pigment epithelial (RPE) cells and vascular endothelial cell lines(VEC304). Methods Human RPE cells and VEC304 were cultured and treated in different human vitreous-conditioned medium (VCM) with or without serum, vitreous volume concentrations of VCM were 1∶8, 1∶4 and 1∶2. Cells proliferation was assayed by tetrazolium (MTT) colorimetry at the 2nd, 4th and 6th day respectively. Results In the presence of serum, 1∶4, 1∶2 VCM had a significantly stimulative effect on RPE cells proliferation compared with control group at the 2nd, 4th, and 6th day retrospectively (P＜0.01), but exerted a bly inhibitory effect on VEC304 proliferation compared with control group at the 2nd, 4th, and 6th day retrospectively (Plt;0.05). In the absence of serum, only 1∶2 VCM had a stimulative effect on RPE cells growth compared with control group at the 2nd day (P＜0.05) and obviously at the 4th and 6th day respectively (P＜0.01). Conclusion Human vitreous fluid influences human RPE cells and VEC304 growth in vitro. This result suggests that vitreous may play different role in proliferative vitreoretinopathy and intraocular neovascular disease. (Chin J Ocul Fundus Dis, 2002, 18: 140-142)