Objective To investigate the ingestion, metabolism and subcellular localization of indocyanine green (ICG) in human retinal epithelial (R PE) cells.Methods RPE cells were incubated with 0.25 mg/ml ICG under the condition of 37oC in the camera. The ICG granule and ultrastructure of RPE cells were observed under the electron microscopy after 1, 4, and 24hour incubation, and the ICG autofluorescence was detected by fluorescence microscopy after the incubation for 1, 2, 4, 8, 12, 24, and 48 hours, respectively. The ab sorbency (A value) of ICG solution was measured at 805 nm with ultraviol et/v isible specrtrometer. The standard curve of concentration of ICG was drawn and the related equation of concentration of ICG and the A value was calculated. After being incubated for 1, 2, 4, 8, 12, 24, 48, and 72 hours, respectively, the A value of supernatant fluid was calculated according to the equation. Aft er incubated with ICG for 24 hours, one sample was observed under electron microscope and fluorescence microscope per week to evaluate the metabolizable period of ICG .Results ICG granules were distributed evenly after entering the RPE cells. After incubated with 0.25 mg/ml ICG for 24 hours, no significant change of the ultrastructure of the RPE cells was found. ICG granules accu mulated in the cells as the time goes by and reached the peak after 24 hours, and then they decreased because of the slowdown of the metabolism. Few ICG was still remained in the cells 1 week later Conclusions RPE cells may take in ICG actively. ICG metabolizable period in RPE cells is long, which may be one of the mechanisms of the toxicity of ICG to the retina in the vitreous operation.(Chin J Ocul Fundus Dis，2004，20：179-181)
Objective To establish a method for primary culture of iris pigment epithelial cells(IPE). MethodsEnzyme-Assisted microdissection was used to isolate and cultivate the IPE cells.An identification was made with microscopic and immunohistochemical observations.Results IPE were successfully sultured and showed on differences with RPE in primary culture and subculture.ConclusionEnzyme-Assisted microdissection is a reliable and quick method for the isolation of IPE.
Objective To investigate the inhibitive effect of E2F decoy oligodeoxynucleotides (E2F decoy ODNs) on cultured human retinal pigment epithelial (HRPE) cells.Methods E2F decoy ODNs or scramble decoy ODNs at varied concentrations were put into the HRPE cells mediated by lipofectamineTM2000. The proliferative activity of HRPE was detected by methythiazolyl-terazollium assay, and the competitive combinative activity of E2F decoy ODNs and transcription factor E2F was detected by electrophoresis mobility-shift assay. Results The proliferation of HRPE was inhibited markedly by E2F decoy ODNs at the concentration of 0.2 μmol/L (P=0.002) in a dose-dependent manner but not by scrambled decoy. The results of electrophoresis mobility-shift assay showed that the combinative activity of transcription factor E2F was abolished completely by E2F decoy ODNs. Conclusions E2F decoy ODNs may sequence-specifically inhibit the combinative activity of transcripti on factor E2F,and inhibit the proliferation of HRPE cells.(Chin J Ocul Fundus Dis，2004，20：182-185)
OBJECTIVE:To investigate the index of the rejection of lJle retinal pigment epithelium(RPE)cells transplantation. METHOD:Allogenic RPE transplantation on rahbits by transcleral technique, the changes of interleukin-2 (IL-2) activity in peripheral blood and the effect of immunoinhibitor (methylprednisonlone)were detected. RESLILTS:In the group of simple transplantation,the IL-2 activity in peripheral blood begin to rise in the first day after operation. The peak value occured in the third day,and is still much higher than that of the control group in the 14th day,whereas in the group treated with immunoinhibitor ,there was no obvious difference in the first day after operatlon,in the third day,the IL-2 activity rises slightly,and returned to normal level in the 7th day. CONCLUSION: After RPE transplantation, the level of IL-2 activity in peripheral blood might serve as an important index to determining and detecting the rejective response. （Chin J Ocul Fundus Dis,1996,12: 239-241）
Objective To explore a better method in obtaining iris pigment epithelium(IPE) specimen for autologous transplantation in rabbits． Methods IPE was obtained from 20 black rabbits with method A，i.e.surgical peripheral iridectomy at 12:00 position obtaining a triangle iris tissue with the hemline of 4-5 mm in left eyes，and method B,i.e.surgical peripheral iridectomy at 11:00 and 1:00 positions obtaining two triangle iris tissues with the hemlines of 2-2.5 mm in right eyes . The IP E cells were isolated precisely with enzyme microdissection-enzyme isolation method, cultured in vitro, observed with light and electronic microscope, and ident ified with immunocytochemical staining.ResultsThe success ra te of cells culture were 65% for method A and 95% for method B. After 3-4 generations of culturing，the amount of IPE cells was enough for transplantation, and most of the functions of primary clutured IPE cells were kept still. Viability of IPE cells was 85%-93%. Conclusion The success rate of cells culture for method B is higher than that for method A. The third generation of cultured cells is available for autologous transplantation.(Chin J Ocul Fundus Dis，2003，19：201-268)
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)
Purpose:To evaluate the function of gap junction-mediated intercellular communication in cultured cells of retinal pigment epithelial(RPE) cells from porcine eyes. Methods:The cultured RPE cells were previously stained by a fluorescent probe 5, 6-carboxy fluorescein diacetate (CFDA) ,and then photobleach the fluorescent molecule in chosed cells. Using laser scanning confocal microscope (LSCM)to observe fluorescence recovery rate of the RPE cells which located in different condition. The function of gap junction communication was evaluated according to the fluorescence recovery rate. Results:The fluorescence recovered after photobleached and the fluorescent density of cells which touching to them descend. The recovery rate per minnte of the cells which the cell number it adjacent to was 1,2 and 3 respectively was 1. 997plusmn;0. 665, 4. 378plusmn;0. 811 and 8. 736plusmn;2. 084. Conclusion:The cultured porcine RPE cells have the function of gap junction communication,and its function proportion is associate to its adjoining cells number. （Chin J Ocul Fundus Dis,1996,12: 41-42）
PURPOSE:In search of the mechanism for photic retinal injury. METHODS:A visible light damage model was established in the primary cultured healthy,adult human RPE cells by using intense fluorescence light (2 400 Lx). RESULTS:Electron microscopy revealed swelling of the mitochondria and obscurity of nuclear membranous structure in the light damaged cells. The decrease or dissolution of organelle,vacuolization of cytoplasm and myelinic degeneration were found in some severely damaged cells. The level of intracellular SOD was decreased to 41% of that of the controls (P＜0.05). CONCLUSION:The structure of the RPE was damaged by the light radiation and the level of intracellular SOD was decreased. These suggested the light damage might be associated with the production of free radicals and the lipid perioxide reaction in membranous structure of cell. （Chin J Ocul Fundus Dis,1996,12: 174-175 ）
Objective To observe the effect of exogenous basic fibrob last growth factor (bFGF) on apoptosis of cultured human retinal pigment epithelial (RPE) cells exposed to visible light,and determine the role of bFGF, fibroblast growth factor receptor 1 (FGFR1),bcl-2 and caspase-3. Methods 2000±500) lx cold white light was used. Exogenous bFGF was utilized during culture. Annexin annexin V-fluoresce in isothiocyanate/propidium iodium (V-FITC/PI) labeling,flow cytometry, Immunocytochemical staining, enzyme associated absorb examing and reverse transcriptional polymerase chain reaction (RT-PCR) were used to determine the apoptosis, the expression levels of bFGF, FGFR1, bcl-2, as well as the activity of caspase-3. Results No protective effect of bFGF was observed under the concentration 5 ng/ml.A significant inhibition of apoptosis was found in 10 ng/ml and 20 ng/ml groups （P＜0.05）. The upregulation of bcl-2 was observed in bFGF (10 ng/ml, 20 ng/ml) protreated groups（P＜0.01）.Compared to no light exposure group,all light exposure groups (including bFGF pro-treated) had higher endogenous bFGF and FGFR1 levels （P ＜0.05）, and the increase was concentration dependent.The bFGF and FGFR1 levels were higher in exogenous bFGF applied (gt;5 ng/ml) groups than light exposure groups（P＜0.05）. The caspase-3 activity was significantly inhibited in bFGF (10 ng/ml) pro-treated groups. Conclusions Human RPE cells exposed to visible light were rescued by application of exogenous bFGF in vitro.The probable protective mechanism of bFGF partly is directly binding to FGFR1 or potentiating endogenous bFGF autocrine loop,to upregulate bcl-2 and to inhibit caspase-3 activation. (Chin J Ocul Fundus Dis，2003，19：24-28)
Objective To investigate the effects of platelet-derived growth factor(PDGF) on the expression of α-smooth muscle actin(α-SMA) of cultured human retinal pigment epithelium cells(RPE). Methods Cultured human RPE cells of the 4-6 th passages were divided into two groups: Delbecco′s modified Eagle′s medium (DMEM) and 2%DMEM (20 g/L foeta calf serum+DMEM). PDGF (0,1,50 ng/ml) was added to medium.The expression of α-SMA was detected and quantitatively analyzed by image process of immunofluorescence.Results PDGF stimulated the expression of α-SMA of human RPE cells.In group of DMEM, The rate of RPE of α-SMA expression was 40%-50% and the intension of fluorescence was 8.08 without PDGF. After stimulated by PDGF(1 ng/ml,50 ng/ml), the rates were 80% and 90% respectively, and the intension of fluorescence were 12.35 and 17.23. In 2%DMEM group, The rates of RPE of α-SMA expression were 85% without PDGF, and 95% ,100% respectively treated with PDGF (1 ng/ml,50 ng/ml). The intension of fluorescence was 14.79 without PDGF, and after stimulated by PDGF, they were 16.28 at 1 ng/ml and 21.36 at 50 ng/ml，which was 2 .7 times ber than that in DMEM group without PDGF. Conclusion PDGF could stimulate RPE cells to express α-SMA. (Chin J Ocul Fundus Dis，2003，19：201-268)