Objective:To study the effects of growth factor on the proliferation of the cultured huamn retinal glial cells. Methods:EGF(0.5～100.0ng/ml) and NGF (0.5～10.0ng/ml) were added to cultures of human retinal glial cells and the proliferation rates of the cells were measured by MTT method. Results:EGF at a dosage ranging from 0.5ng/ml to 100.0ng/ml and NGF (0.05～10.0ng/ml) stimulated the cellular proliferation effectively with their EC 50 of 17ng/ml and 0.7 ng/ml respectively. Conclusion:Both EGF and NGF NGF had an effective stimulation on human retinal glial cell proliferation.They may play a role in the formation of PVR. (Chin J Ocul Fundus Dis,1998,14:33-34)
Objective To determine the effect of thiazolidinediones (TZDs) on early retinopathy in rats with experimental diabetes. Methods In 40 rats, diabetic models were set up in 36 by one-off intraperitoneal injection with streptozotocin (STZ), and other 4 were in the normal control group. Twenty-four diabetic rats with the disease-duration of more than 6 months underwent intravitreous injection (with rosiglitazone or pioglitazone in 10 rats, respectively), and the rest 4 rats werenprime;t injected with drugs as the diabetic positive control group. Immunohistochemical treptomycin-avidin-biotin-complex (SABC) method, in situ hybridization of retinal vascular endothelial growth factor (VEGF) mRNA, and TdT-dUTP terminal nick-end labelling (TUNEL) were performed on the ocular paraffin section to detect the cellular apoptosis. The difference of VEGF expression and cellular apoptosis between TZDs and control group was observed and analyzed. Results The results of immunohistochemical staining and hybridization in situ were negative in the normal control group. The positive expression rate of VEGF was lower in rosiglitazone and pioglitazone group than which in the diabetic positive control group, and there was no obvious differences of positive expression of VEGF mRNA and cellular apoptosis between the 2 groups. Conclusion TZDs (rosiglitazone and pioglitazone) may inhibit the positive expression of VEGF protein in retina of STZ-induced diabetic rats to some extent, but not affect the growth of VEGF in retina. (Chin J Ocul Fundus Dis, 2006, 22: 7-10)
Objective To observe the expression of p53, bcl-2 genes, vascular endothelial cell growth factor（VEGF）, basic fibroblast growth factor（bFGF）, insulin-like growth factor-I （IGF-I）, and the receptors of these factors of retinal vascular endothelial cells (VECs) of 1- to 20-week diabetic rats, and the relationship between the expressions and cell cycle arrest.Methods Retinal sections of diabetic rats induced by alloxan were immunohistochemically stained and observed by light microscopy (LM) and electron microscopy (EM). Dot blotting and Western blotting were used to determine the expression of mRNA, proteins of p53 and bcl-2. Results Under LM, immunohistochemical positive expression of p53 and bcl-2 were found on the vessels of ganglion cell layer and inner nuclear layer of retinae of 8- to 20-week diabetic rats; under EM, these substances were observed depositing in VECs. The retinal VECs also expressed VEGF, bFGF, IGF-I and their receptors. There was no positive expression of other cell types in these retinae, all cell types of retinae in control group, or all cells of retinae of diabetic rats with the course of disease of 1 to 6 weeks. The result of dot blotting revealed that retinal tissue of 20-week diabetic rat expressed p53 and bcl-2 mRNA, and the result of Western blotting revealed that they also expressed p53 and bcl-2 proteins. But retinal tissues of control group did not. Positive expression of bax was not found in the retinae in control group or 1- to 20-week diabetic rats. Conclusion p53, bcl-2 may introduce cell cycle arrest of VECs of retinae in 8- to 20-week diabetic rats. High glucose might stimulate the expression of VEGF, bFGF, IGF-I and their receptors, and the growth factors may keep VECs surviving by self-secretion. (Chin J Ocul Fundus Dis，2003，19：29-33)
Objective To investigate the effect of batroxobin on the blood-retinal barrier (BRB) and vascular endothelial growth factors (VEGF) in diabetic rats. Methods Sixty Sprague-Dawley rats were used to establish diabetic models by intraperitoneal injecting with streptozotocin (60 mg/kg), and were divided into 3 groups: diabetic group (n=20), batroxobin (40 mg/kg) group (n=20) and batroxobin (20 mg/kg) group (n=20). Twenty-five else rats were in control group. All of the rats were executed 7 days later. The function of BRB was observed by Evans blue method. Results concentration of VEGF protein was detected by enzyme-linked immunoabsorbert assay (ELISA). The results of each group were compared. Results The content of BRB leaked into retina was obvious lower in the control group than which in the other 3 diabetic groups（Plt;0.01）. There was no significant difference of the content of Evans blue between the two groups with different dosage of batroxobin (P＞0.05). The content of Evans blue was lower in the 2 diabetic groups with different dosage of batroxobin than which in the control group (Plt;0.05). The content of VEGF in retina was obviously lower in control group and 2 diabetic groups with different dosage of batroxobin than which in the diabetic group (Plt;0.01), and obviously lower in batroxobin (40 mg/kg) diabetic group than which in the control group (P＝0.01). The content of VEGF in control group and batroxobin (20 mg/kg) diabetic group (P＝0.06) didnprime;t differ much, which occurred similarly in batroxobin diabetic groups with different dosage (P＝0.78). Conclusions Batroxobin may alleviate the damage of function of BRB in diabetic rats and reduce the expression of VEGF, which suggests that batroxobin can protect the function of BRB to a certain extent. (Chin J Ocul Fundus Dis, 2006, 22: 16-19)
Objective To observe whether apoptosis was involved in cells of aspiration fluid from vitrectomy for proliferative vitreoretinopathy(PVR),and whether there was an association with expression of Fas antigen(Fas )and Fas ligand (FasL). Methods Cytocentrifuge slides of 11 fresh vitreous specimenｓ of PVR were prepared to be stained by TUNEL met hod for detection of apoptosis and by immunohistochemical technique for detection of Fas,FasL，and cytokeratin (CK),a cell-type specific antigen. Results Fas and FasL were expressed in normal human retina.Fas,FasL,CK，and apoptosis were found in all preparations.TUNEL-positive cells were 20.53% in total cells.70.35%,51.58%，and 82.97% of cells highly expressed Fas,FasL，and CK,respectively.The linear correlation coefficient of Fas and apoptosis was 0.99(Plt;0．001). Conclusion Vitrectomy specimens of PVR showed expression of Fas，FasL，and apoptosis.Prominent Fas and FasL expressions may be associated with apoptosis of proliferating retinal pigment epithelial cells in the vitreous of PVR. (Chin J Ocul Fundus Dis,1999,15:78-80)
Objective To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells. Methods Cultured hRPE-19 cells (3rd - 6th generations) were used in this experiment. hRPE-19 cells were divided into three groups including the control group, the hypoxia experimental group (100 μmol/L CoCl2) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia). After culturing for 4, 8, 12 and 24 hours, the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction, and the protein level of Shh and VEGF in the supernatants were measure by enzyme-linked immunosorbent assay. The relationship between the expression of Shh and VEGF was analyzed by Pearson correlation analysis. Results The control group expressed low levels of Shh and VEGF mRNA/protein. The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364, 183.732, 77.456, 91.572; P＜0.01). The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745, 121.834; P＜0.01). In the hypoxia experimental group, the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942, P＜0.05); and the expression of VEGF and Shh mRNA was positively correlated (r=0.970, P＜0.01). However, there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915, P＞0.05). Conclusion There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.
Objective To investigate the effects of epidermal growth factor (EGF)，fibroblast growth factor(FGF), and bovine serum on proliferation and apoptosis of the cultured fetal human retinal cells.Methods EGF and FGF were added or not to the medium of fetal human retinal cells cultured by bovine serum in vitro. The number of cells, bromodeoxyuridine(BrdU) incorporation and Tdt-mediated dUTP nick end labelling(TUNEL) were detected to determine the proliferation and apoptosis. Immunohistochemical staining of neuron specific enolase(NSE), Thy1.1, glial fibrillary acidic protein(GFAP) and scan electromicroscopy were performed to identify cell components. The expression of transcription factor c-fos, c-jun and apoptosis regulation factor bcl-2 and Bax were examined by immunohistochemical staining to explore the underlying mechanism.Results The increased number of NSE and Thy1.1 positive cells and BrdU incorporation, and decreased apoptotic cells were found in the groups treated with EGF and FGF. Meanwhile, the up-regulation of c-fos, c-jun and bcl-2 were also found. Conclusion EGF and FGF can promote the survival and proliferation of cultured retinal cells by up-regulating the expression of c-fos, c-jun and bcl-2. (Chin J Ocul Fundus Dis，2003，19：113-116)
ObjectiveTo study the changes and correlation of cytokines in aqueous humor before and after intravitreal injection of conbercept (IVC) treatment in patients with proliferative diabetic retinopathy (PDR).MethodsA prospective clinical study. From March to December 2019, 36 patients (42 eyes) of PDR patients treated with IVC combined with pars plana vitrectomy (PPV) (the observation group) and 27 patients (31 eyes) underwent cataract surgery in the same period (control group) in Department of Ophthalmology of the First Affiliated Hospital of Guangzhou University of Chinese Medicine were included in this study. Before PPV 5-7 days, IVC treatment was performed, and the aqueous humor were extracted during IVC and second-stage PPV in the observation group. The aqueous humor was extracted during cataract surgery in the control group. Luminex assay was used to detect VEGF-A, placental growth factor (PLGF), platelet-derived growth factor-AA (PDGF-AA), platelet-derived growth factor-BB (PDGF-BB), angiopoietin-like protein 4 (ANGPTL4), IL-6, IL-8, IL-1β, monocyte chemoattractant protein-1 (MCP-1), intercellular adhesion molecule-1 (ICAM-1), tumor necrosis factor-α (TNF-α) cytokine expression. For normally distributed data, the independent sample t test was used for comparison between two independent samples; for non-normally distributed data, the Wilcoxon rank sum test was used for comparison between two independent samples. The correlation analysis used Spearman rank correlation test.ResultsBefore IVC treatment, the concentrations of VEGF-A, PLGF, PDGF-AA, ANGPTL4, IL-6, IL-8, MCP-1 and ICAM-1 in the aqueous humor of PDR patients were significantly higher than those in the control group (P＜0.05). After IVC treatment, the concentration of VEGF-A in the aqueous humor was significantly lower than that before treatment, and the concentrations of ANGPTL4 and IL-8 were significantly higher than those before treatment (P＜0.05). There were no significant differences in the concentrations of PLGF, PDGF-AA, PDGF-BB, IL-6, IL-1β, MCP-1, ICAM-1 and TNF-α before and after IVC treatment (P＞0.05). Before IVC treatment, the concentration of VEGF-A was positively correlated with PLGF, PDGF-AA, PDGF-BB, ANGPTL4, IL-6, IL-8, MCP-1 and TNF-α (P＜0.05).ConclusionsIVC treatment can reduce the concentration of VEGF-A and increase the concentrations of ANGPTL4 and IL-8 in aqueous humor in PDR patients before PPV.
ObjectiveTo observe the expression of Rap1, guanosine triphosphate-Rap1 (GTP-Rap1), vascular endothelial growth factor (VEGF) and β-catenin in experimental choroidal neovascularization (CNV).MethodsForty-two brown Norwegian rats were randomly divided into a blank control group (7 rats) and a model group (35 rats). Both eyes were enrolled. The CNV model was established by holmium ion laser photocoagulation in the model group. At 3, 7, 14, 21, and 28 days after photocoagulation, fluorescein fundus angiography (FFA) and choroidal vascular smear were performed to observe the degree of fluorescein leakage and CNV area in rats; Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to detect the expression of Rap1, GTP-Rap1, VEGF, β-catenin and mRNA in CNV.ResultsThe results of FFA examination showed that a large disc-shaped fluorescein leaked in the photo-condensation spot 14 days after photocoagulation. Laser confocal microscopy showed that compared with 7 days after photocoagulation, CNV area increased at 14, 21, 28 days after photocoagulation, and the difference were statistically significant (t=3.725, 5.532, 3.605;P＜0.05). Western blot showed that there was no significant difference in the relative expression of Rap1 protein in CNV at different time points after photocoagulation between the two groups (P=0.156). Compared with the blank control group, the relative expression of GTP-Rap1 protein was significantly decreased, the relative expression of VEGF and β-catenin protein were significantly increased in the model group (P=0.000). The results of RT-PCR showed that there was no significant difference in the relative expression of Rap1 mRNA at different time points after photocoagulation between the two groups (P=0.645), but there were significant difference in the relative expression of β-catenin mRNA (P=0.000). At 7, 14, 21 and 28 days after photocoagulation, there were significant difference in the relative expression of GTP-Rap1 and VEGF mRNA between the two groups (P=0.000).ConclusionsThe expression of GTP-Rap1 in experimental CNV is significantly lower than that in normal rats.