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 determine the expression of the growth factors and the receptors related to angiogenesis in the intraocular tissues incarcerating in the sclerotomy sites. Methods Ten specimens from prolapsing intraocular tissues in sclerotomy sites during vitrectomy were obtained and serially sectioned in cryostate and were stained with a group of polyclonal antibodies against vascular endothelial growth factor(VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-A(PDGF-A) and transforming growth factor-β1(TGF-β1) as well as their receptors by using a streptavidin peroxidase system. Results The tissues prolapsed from the sclerotomy sites were identified as retina(3 cases), vitreous tissues(3 cases), degenerated red blood cell components(2 cases), ciliary body(one case) and fibrous tissue(one case). All specimens expressed VEGF and bFGF as well as their receptors. PDGF-A, TGF-β1 and their receptors expressed in the most of specimens. The positive cells included retinal cells, ciliary non-pigmented epithelial cells and pigmented epithelial cells, fibrous cells and the cells in vitreous. Conclusions The intraocular tissues incarcerated in the sclerotomy entries express the growth factors and receptors related to angiogenesis. This might be one of the potential factors of developing anterior proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis, 2002, 18: 34-37)
Objective To detect the concentration of vascular endothelial growth factor (VEGF) in plasma and intraocular liquid (aqueous humor and vitreous body) in patients with deabetic retinopathy (DR) and the role VEGF plays in the development of DR. Methods The concentrations of VEGF in plasma, aqueous humor and vitreous body in DR and normal group were detected by ELISA. Results The concentration of VEGF in plasma was (34.47plusmn;1.76) pg/ml in non-DR group, (53.93plusmn;3.08) pg/ml in single DR group, (53.36plusmn;3.28) pg/ml in proliferative DR group, and (178.30plusmn;10.13) pg/ml in control group. There was no significant difference in the normal and the experimental groups (P＜0.05). The concentration of VEGF in aqueous humor was (184.8plusmn;12.60) pg/ml in proliferative DR group and (90.06plusmn;8.32) pg/ml in the control group, and there was significant difference between them (P＜0.05). The concentration of VEGF in vitreous body was (741.70plusmn;92.02) pg/ml in proliferative DR group and (94.38plusmn;21.21) pg/ml in the control group, and there was significantdifference between them (P＜0.05). There was no correlation of VEGF concentration in plasma and that in aqueous humor and vitreous respectively(P＞0.05), and positive correlation of VEGF concentration was found in vitreous body and HbA1c (r=0.9067,P＜0.01). Conclusions Concentration of VEGF in plasma in patients with DR is lower than that in the normal persons,but not correlated with the concentration of VEGF in aqueous humor and vitreousbody. The concentration of VEGF in aqueous humor and vitreous body increase in patients with proliferative DR, and the increase in vitreous body and the value of HbA1c of the patients correlate. (Chin J Ocul Fundus Dis，2004，20：343-345)
PURPOSE:To approach the establishment of t be optimal method for determining the intracellular free Ca2+ concentration[Ca2+]i of dissociated newborn rabbit retina cells by using fluorescent indicator-Fura-2/AM. METHODS:Trypsin was employed to prepare the retlna cell suspensions which were then loaded with Fura-2/AM followed by fluorescence determination. RESULTS:The cellular viability rate of retina cell suspensiotls prepared by 0.05% trypsin 10 minutes at 37deg;C was over 90%. Loading the retina cell suspensions with Fura-2/AM 40 minutes at 37deg;C and then measurlng the fluorescent intensity of the suspensions within 30 minutes were proved to be the optimum. CONCLUSIONS:The resting [Ca2+]i of retina cell suspension was (223plusmn;27)nmol/L whlch was within the expected range of [Ca2+]i level. 25mmoI/L and S0mmol/L K+ increased the [Ca2+Ji 59% and 148% respectively. These results indicate that the preparation of retina cell suspensions and the method of [Ca2+Ji determination are reliable and feasible. （Chin J Ocul Fundus Dis,1996,12: 108-110 ）
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)
Objective To describe cultured human retinal pigment epithelial (RPE) cells transdifferentiation and investigate the effects of human vitreous fluid on the morphologic and cytoskeleton changes of RPE cells in vitro. Methods Cytoskeleton characteristics in the 2nd, 5th, 8th passage of RPE cells in normal culture, which included cytokeratin 18 (CK18) and α-smooth muscle actin (α-SMA) were analyzed by Western blot. RPE cells were cultured in human vitreous-conditioned medium (VCM) at the concentration of 1∶4 for 6 days, morphologic changes were examined by light and electron microscopy, and cytoskeleton characteristics were analyzed by imunocytochemistry and Western blot. Results During culture in vitro, RPE cells lost epithelial characteristics and aquired fibroblast-like phenotype. The expression of CK18 was the highest at the 5th passage, and it decreased in the following passage, but α-SMA increased gradually. The morphologic transdifferentiation from epithelial to fibroblast-like cells of RPE was accelerated by VCM. Ultrastructural changes such as decreased microvilli and gradually increased rough endoplasmic reticulum and Golgi complex were found during the cultivation. CK18 produced by RPE cells decreased in VMC (P＜0.05), and α-SMA increased (P＜0.01). Conclusion Morphologic changes in epithelialmesenchymal transdifferenetiation of RPE cells are stimulated by VCM and accomplied by the shift of cytoskeleton proteins, The results imply that cells migration may be decreased and contraction may be enhanced in VCM. It may suggest that vitreous accelerates the pathogenesis of PVR and RPE cells play an important role. (Chin J Ocul Fundus Dis, 2002, 18: 289-292)
PURPOSE:To investigate mitochondrial DNA(mtDNA) of Leber's hereditary optic neuropathy(LHON). METHODS:Polymerase chain reaction(PCR)method was used to analyse mtDNA of 11 patients in a pedigree with LHON and 4 control subjects from none LHON pedigree. RESULTS:There was a loss of a restriction site for the restriction endonuclease SfaN.Ⅰin Ihe Patients with LHON. In this pedigree,maternal lineage was regarded a carrier of the pathogenic gene. CONCLUSIONS:The patients with Leber's hereditary optic neuropathy have a point mutation in mtDNA,which results in loss ol SfaN I endonuclease restriction site .and this change is one of mechanisms inducing this disaese. （Chin J Ocul Fundus Dis,1997,13: 27-29）
Objective To verify the effect of Evans blue dye on determining the retina blood vessel leakage． Methods Male Sprague-Dawley rats were used in this study． The VEGF induced retinal blood vessel leakage was checked with Evans blue dye． Then the bloodretina barrier breakdown of 1 week diabetic animals was quantified with Evans blue．The dye was extracted from retina by formamide and the extraction was checked with spect rophotometer． Evans blue leakage was normalized against wet or dry retina weight． Results The retinal Evans blue content of eyes treated with VEGF was remarkably higher than that of the controls (n=17 ,Plt;0．0001)． And the eyes of 1 week diabetic duration animals had more Evans blue dye than that of the normal controls (Plt;0．05)． Conclusion Evans blue dye is a sensitive tracer in quantitatively diagnosing the blood retina barrier breakdown． (Chin J Ocul Fundus Dis, 2001,17:221-223)