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 detect the apoptosis of vascular endothelial cells and retinal pigment epithelial (RPE) cells in vitro induced by verteporfin-photodynamic therapy. Methods Cultured vascular endothelial cells and human RPE cells were incubated with verteporfin at a concentration of 1.0 mu;g/ml which was equivalent to the initial plasma level of verteporfin in clinical therapy. Each kind of cells were divided into 6 groups according to different time of incubation: 0, 5, 15, 30, 60, and 120 minutes group. After incubated, the cells were illuminated by the laser light with the maximum wavelength of absorption of verteporfin (wavelength: 689 nm, power density: 600 mW/cm2) with the power of 2.4 J/cm 2for 83 seconds. The percentage of cellular apoptosis was measured by flow cytometry 3 hours after PDT, and the measurement was repeated thrice. Results The proportion of cellular apoptosis 3 hours after PDT were 0.01plusmn;0.01, 0.25plusmn;0.02, 0.32plusmn;0.02, 0.41plusmn;0.04, 0.49plusmn;0.03 and 0.61plusmn;0.02, respectively in 0-120 minutes group of vascular endothelial cells; and 0.02plusmn;0.01, 0.22plusmn;0.01, 0.31plusmn;0.02, 0.38plusmn;0.03, 0.47plusmn;0.05 and 0.58plusmn;0.03 respectively in 0-120 minutes group of RPE cells. The proportion of cellular apoptosis of both kinds of the cells increased as the incubation time was prolonged. There was no significant difference of the percentage of cellular apoptosis between the accordant time groups in the two kinds of cells (P＞0.05). Conclusions Cellular apoptosis can be quickly induced by verteporfin-PDT both in human vascular endothelial cells and RPE cells; under the same condition in vitro, PDT has no obvious selection for the apoptosis of the two kinds of cells. (Chin J Ocul Fundus Dis, 2006, 22: 253-255)
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)
Purpose To investigate retinoic acid (RA) induced apoptosis in retinal pigment epithelial (RPE) cells. Methods 10-5、10-6、10-7 mol/L were added to cultured PRE cells.Aridine orange fluorescence and TdT-mediated dUTP nick end labelling(TUNEL) techniques were used to observe apoptotic changes. Resultss 10-5、10-6、10-7 mol/L RA induced apoptosis in RPE cells.Cell shringkage,chromatin condensation and nuclear DNA fragmentation of RPE cells were observed by TUNEL technique.When 10-7、10-6、10-5mol/L RA treated RPE cells for 5 days,apoptotic index(AI)was 36.9%、4409% and 61.4% respectively,and 48.0%、59.9%、74.2% for 6 days.At the same concentration of RA,AI increased when time prolonged．At the same day,AI increased when the concentration of RA rose.There was significant difference in the results(Plt;0.05). Conclusion Our results showed that RA-induced apoptosis in RPE cells was detected with a good dose and time response. (Chin J Ocul Fundus Dis,1998,14:153-155)
Objective To investigate the effect and mechanism of Ginkgo biloba extract EGb761 on protein expression in lightdamaged retinal pigment epithelial (RPE) cells. Methods The human RPE cells (ARPE19) were divided into normal control group, light damage group and EGb761 treatment group; the cells of latter 2 groups were exposed to the cold white light ［(2200 ± 300) lx］ to induce light damage responses. The lightdamaged RPE cells were treated with or without EGb761 (100 g/ml). The soluble protein of those cells were extracted and separated by twodimension electrophoresis and stained by silverstaining. Different proteins in the gel were analyzed by ImageMaster and identified by MALDITOFMS, and were further analyzed by mass spectrometry and bioinformatics.Results ImageMaster and MALDITOFMS identified 25, 33 and 11 different proteins between light damage group and EGb761 treatment group, between normal control and light damage group, between normal control and EGb761 treatment group of RPE cells respectively. Mass spectrometry and bioinformatics analysis successfully identified 16 proteins, including metabolic enzymes, cytoskeleton proteins, antioxidation protein and other types of proteins expressed differentially．Conclusion Protein expression profiles are different between normal control group, light damage group and Ginkgo biloba extract treatment group of RPE cells. The mechanism of protective effect of EGb761 may involve cathepsin B, heat shock protein, cytochrome C reductase, and other proteins.
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)
ObjectiveTo investigate the cytotoxicity of triamcinolone acetonide (TA) on cultured human retinal pigment epithelial (RPE) cells.MethodsEffect of TA with different concentraion (0.4, 02, 01, 0.05, and 0.025 mg/ml) on the proliferation of RPE cells was detected by methyl thiazolyl tetrazolial (MTT) assay; The changes of cellular cycle treated by TA with the drug concentration of IC50 for 72 hours were measured by flow cytometry (FCM) ananlysis, and the morphological and ultrastructural changes of the cells were observed by phasecontrast microscopy and transmission electron microscopy.ResultsWith the concentration of 0.4-0.025 mg/ml, TA inhibited the growth of RPE cells obviously in a dose-dependent manner compared with the control (P＜0.05), and the cells in S phase treated with TA decreased 10.6% compared with the control (P＜0.05). Under the light microscope, sparse cells with irregular configuration and many prominences and cellular vacuoles in TA group can be seen. The results of transmission electron microscopy showed asymmetrically distributed cellular chromatin was, cavitated cytoplasm, and even some necrotic cells.ConclusionTA can inhibite the S-phase karyomitosis of RPE and inhibite the cellular proliferation; and destroye the cellular structure and lead to the necrosis, which indicates the cytotoxicity of TA on RPE.(Chin J Ocul Fundus Dis, 2005,21:233-236)
OBJECTIVE:To observe the effect of dexamethasone to intracellular free Ca2+ of frozen RPE cells. METHODS:The cultured human RPE cells were frozen for 30s at --70deg;C. The RPE cells were loaded with Fura-2/AM and analyzed using a digital imaging microscopy system,the effect of dexamethasone to intracellular free Ca2+ was measured at a serial concentration of 40, 60,100,150,200mu;g/ml. RESULTS:The concentration of intracellular free Ca in frozen human RPE cells was increased to 18.6%~29.8% by dexamethasone at concenlration of 40mu;g/ml~60mu;g/ml,while was decreased to 28.4%~35.2% at 150mu;g/ml~200mu;g/ml. CONCLUSIONS:Effect of dexamethasone showed two aspects of effect to frozen cultured human RPE ceils,that it was inhibitor at high concentration and stimulator at low concentration （Chin J Ocul Fundus Dis,1997,13: 86-88）
Objective To investigate the modulating effect of transforming growth factor beta;2 (TGFbeta;2) and extracellular matrix (ECM) on the transdifferentiation of human fetal RPE (hfRPE) cells into myofibroblast-like cells , and to determine the mechanism of signal transduction. Methods hfRPE cells were cultured on ECM coated or uncoated petri dish with or witho ut TGFbeta;2 in the medium. The expression of alpha;-smooth muscle actin (alpha;-SMA) were detected by immunocytochemistry examination, flow cytometry and Western blotting via calphostin C, genistein, PD98059, and Wortmannin. Results After cultured on ECM coated petri dish with TGFbeta;2 in the medium,there were obvious morphological changes of hfRPE cells including cellular elongating and appearing of actin microfilaments. The results of flow cytometry and immunocytochemistry examination showed that expression of alpha;-SMA obviously increased after TGFbeta;2 was added in the medium in a dose-dependent manner. Compared with which of hfRPE cells cultured on the uncoated surface of culture plates, the total mean fluore scence intensity (TMFI) of hfRPE cells cultured on FN-coated surface increased (38.01plusmn;1.14)% when the stimulation concentration of TGFbeta;2 was 50ng/ml(Plt;0.05). Western blotting further confirmed the effects. The changes mentioned above could be inhibited mostly by protein kinase C (PKC) and calphostin C (10 nmol/L)(Plt;0.01). Conclusion TGFbeta;2 may induce the transdifferentiation of hfRPE cells into myofibroblast-like cells in a dose dependent manner, which could be intensified by FN. These mediated effects of TGFbeta;2 and ECM may act via the PKC signal transduction pathway. (Chin J Ocul Fundus Dis, 2006, 22: 328-332)