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)
ObjectiveTo observe the expression of vascular endothelial growth inhibitor (VEGI, TL1A), vascular endothelial growth factor (VEGF), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in diabetes rats' serum, vitreous and retina, and discuss the role of VEGI in the pathogenesis of diabetic retinopathy (DR). MethodsA total of p70 adult male Wistar rats were randomly divided into 4 groups, the control group (10 rats), the diabetes mellitus (DM) 1 month group (20 rats), the DM 3 month group (20 rats) and the DM 6 month group (20 rats). Cytokines of serum and vitreous were determined by enzyme-linked immunosorbent assay (ELISA), and the concentrations of the cytokines in the retina were determined by immunohistochemistry on paraffin retinal sections. Hematoxylin-eosin (HE) staining of retina was used to estimate the pathological change of DR. The results were analyzed by one-way analysis of variances, independent samples t-test and LSD test. ResultsThe serum TL1A levels of the control group, the DM 1 month group, the DM 3 month group and the DM 6 month group rats were (92.09±2.05), (118.36±8.30), (85.90±7.51) and (78.90±4.88) ng/L respectively, the level of TL1A in serum of the DM 1 month group, the DM 3 month group and the DM 6 month group were significantly lower than that of the control group (F=77.405, P < 0.05). The concentration of serum TNF-α and IL-1β increased after DM model was established (F=3.508, 15.416; P < 0.05); the VEGF level in serum showed no difference between the groups (F=1.242, P > 0.05). The vitreous TL1A levels of the control group, the DM 1 month group, the DM 3 month group and the DM 6 month group were (91.50±8.18), (67.03±6.74), (47.44±4.92) and (46.01±4.62) ng/L respectively, every DM groups showed significant difference with the control group (F=114.777, P < 0.05); VEGF level in vitreous increased from 1 month after DM model was established (F=8.816, P < 0.05); TNF-α and IL-1β level in vitreous also showed an upward tendency (F=4.392, 3.635; P < 0.05). Paraffin section immunohistochemistry showed that the absorbance (also called optical density) of TL1A of the DM 1 month group and the DM 3 month group were significantly lower than that of the control group (t=6.851, 6.066; P < 0.05), but the DM 6 month group showed no difference with the control group (t=1.401, P > 0.05); the level of VEGF and TNF-α in DM groups were higher than that of the control group (tVEGF=-4.709, -16.406, -9.228; tTNF-α=-4.703, -6.583, -17.762; P < 0.05); the level of IL-1β were significantly higher in the DM 1 month group and the DM 6 month group (t=-4.108, -3.495; P > 0.05); but the DM 3 month showed no difference with the control group (t=-0.997, P > 0.05). HE staining of retina showed that the retina of the control group and the DM 1 month group had normal retinal structures, the DM 3 month group had retinal edema and disorganization, the DM 6 month group had severe retinal edema, deep stain of ganglion cells, and more neovascularization in inner plexiform layer. ConclusionVEGI is involved in the pathogenesis of DR, and it might interacts with VEGF, TNF-α and IL-1β to affect the development of DR.
Proliferative vitreoretinopathy (PVR) is a common complication and major cause of blindness of ocular trauma. Many cytokines, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), participate in the process of the pathogenesis of traumatic PVR. VEGF competitively inhibits binding of PDGF to its receptor (PDGFRα), enables indirect activation of PDGFRα by non-PDGF ligands, resulting in reduced p53 expression, cell proliferation and migration, which is a key point in the pathogenesis of traumatic PVR.
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.
ObjectiveTo observe the efficacy and safety of individual dose of intravitreal conbercept (IVC) in the treatment of retinopathy of prematurity (ROP) before type 1 threshold.MethodsA retrospective case study. From January to July, 2019, 23 cases (46 eyes) of children with type 1 pre-threshold ROP were included in the study. Among them, 14 cases (28 eyes) were male and 9 cases (18 eyes) were female. The mean gestational age at birth was 28.06±1.73 weeks. The average birth weight was 1.14±0.19 kg. The mean corrected gestational age was 34.38±1.41 weeks at the time of first intravitreal injection of IVC. The axial length (AL) of children was measured by A-mode ultrasound before IVC for the first time. According to the calculation of AL, the corresponding injection dose range was 14.23-16.19, 16.20-17.57, 17.58-18.63 mm and the injection dose of IVC was 0.015, 0.020, 0.025 ml (including IVC was 0.15, 0.20, 0.25 mg, respectively). The first IVC dose was 0.015 ml. On the first day before IVC and on the first and seventh days after IVC, 2 ml of arterial blood was taken from children, serum vascular endothelial growth factor (VEGF) concentration was detected. The follow-up time after treatment was ≥1 year. After one year of follow-up, the effective rate and recurrence rate of IVC for the first time were tested by χ2 tests. The short-term changes of injection times, injection intervals, retinal vascularization time and serum VEGF concentration in children were tested by t test.ResultsRetinal neovascularization subsided and vascular buckling decreased in all eyes. Iris neovascularization subsided, 1-3 weeks after IVC for the first time. Within one year after the first IVC, 16 eyes underwent IVC twice with or without new blood vessels at the junction of the vascular area. The average corrected gestational age was 40.56±3.81 weeks. The injection dose of IVC was 0.015 ml and 0.020 ml for 2 eyes and 14 eyes, respectively.The mean interval from IVC for the first time was 40.89±8.99 days. Of the 16 eyes who underwent IVC twice, 8 eyes showed neovascularization again in the retinal area with or without blood vessels. The average corrected gestational age was 43.00±1.41 weeks. The injection dose of IVC was 0.020 ml and 0.025 ml for 3 eyes and 5 eyes, respectively. The mean interval of the second IVC was 28.60±6.07 days. The mean interval from the first IVC was 69.20±12.40 days. At the end of follow-up, all eyes were treated effectively (100%, 46/46). The mean time of retinal vascularization was 46.31±3.42 weeks. The average number of injections was 1.52±0.76. On the first day before IVC and on the first and seventh days after IVC, the average serum VEGF concentrations were 111.21±148.71, 25.60±27.71 and 42.99±38.01 pg/ml, respectively. Serum VEGF concentration was significantly lower than that before IVC on the 1st and 7th day after IVC (Z=?4.054, ?2.779; P＜0.05). Serum VEGF concentration was higher 7 days after IVC than 1 day after IVC, and the difference was statistically significant (Z=?2.505, P＜0.05). All eyes were not treated by laser photocoagulation or vitrectomy. No eye complications such as lens opacification, endophthalmitis and retinal detachment related to drugs or treatment methods were found in all patients.ConclusionIntravitreal injection of individualized dose of IVC is effective in the treatment of type 1 pre-threshold ROP. Seven days after treatment, serum VEGF concentration of patients’serum decreases.
ObjectiveTo investigate the expression and relation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rats with diabetic retinopathy.MethodFifty-five Wistar rats were randomly divided into the control group (10 rats), and 1, 3, and 5-month-diabetes group (15 rats in each diabetes group), and the diabetic models were set up. The expressions of VEGF and bFGF were detected by situ hybridation and immunohistochemistry on retinal paraffin sections.ResultsThe results of situ hybridation showed that expression of bFGF was found in 3-month-deatbtes group with the percentage of 77.8%, and 88.9% in 5-month-deatbtes group; the positive expression of VEGF was not found in 3-month-deatbtes group but in 5-month-deatbtes group with the percentage of 66.7%. Immunohistochemistry indicated that the positive expression of bFGF started in 3-month-deatbtes group with the percentage of 55.6%, and 88.9% in 5-month-deatbtes group; the percentage of the expression of VEGF was 33.3% in 3-month-deatbtes group and 88.9% in 5-month-deatbtes group.ConclusionThe expression of VEGF occurs after the expression of bFGF in rats with DR.(Chin J Ocul Fundus Dis, 2005,21:37-40)
Objective To observe the expression of the pigment epithelium derived growth factor (PEDF) in retina and the effects of PEDF for vascular endothelial growth factor (VEGF) and retinal microvascular after recombinant adeno-associated virus mediated pigment epithelium derived factor（rAAV-mediated-PEDF）transferred retina of diabetic rats. Methods Male Wister rats were induced diabetes with intraperitoneal injection of streptozocin, then divided into 1 month group (DM1), 3month group(DM3) and 6 month group randomly. In diabetic rats, right eyes were injected rAAV2CMVhPEDF into vitreum as treat group,left eyes were injected into rAAV2-CMV-GFP (1times;1011 v.g/ml) as self-control group. In normal rats, right eyes were punctured but not injected (CONf), left eyes let it be without any disposal. The levels of VEGFmRNA and hPEDFmRNA in retina were evaluated using RT-PCR in different period. The protein levels of VEGF, PEDF within the retina were determined using western blot. The change of retinal capillary were observed through retinal vascular flattening. Result The expression of hPEDFmRNA in retina was enhanced persistence after rAAV2-CMV-hPEDF injected, achieved climax until 6 months. The levels of PEDF were also incerased consecutive, the differences were statistically significant in treatment group compared with own control group （Plt;0.01）. Levels of mRNA and protein of VEGF at different time-points among therapy group were not statistically significant, all obviously higher than normal （Plt;0.05），but all lower obviously than respectively own control group at the same timepoint （Plt;0.01）. The morphology of retinal capillary was not different significant with normal rats in 1 month diabetic rats. Morphology changes of therapy groups were less than those of respective own control group in DM3 and DM6. Conclusion Intravitreous injection rAAV2-CMV-hPEDF can increase expression of mRNA and protein of PEDF，alleviate lesion of retinal microvascular in early period of diabetic rats and supress expression of VEGF in retina of diabetic rats.The regulation occur on mRNA level. （Chin J Ocul Fundus Dis，2008，24：259-264）
Objective To study the effect of serum rich in growth factors (SRGF) derived from plateletrich plasma (PRP) on the biological function of human and rat osteoblast.Methods PRP and platelet-poor plasma (PPP) obtained from healthy human and SD rat were activated by thrombin toget SRGF and serum poor in growth factors (SPGF). The level of TGFβ1 and PDGF-AB in human-SRGF and SPGF were assayed by enzyme-linked immunoassay(ELISA). Rat and human osteoblast were cultured and identified. Rat osteoblasts were treated with 5% rat-SRGF, 5% rat-SPGF and serumfree F12 medium, respectively. And human osteoblast were treated with 5% human-SRGF, 5% human-SPGF and serumfree DMEM. Cellular mitogenic activity was evaluated by thiazoly blue (MTT) colorimetric assay at 24, 48, 72 and 96hours.Results The level of TGF-β1 in human-SRGF was 307.67±35.57 ng/ml, and that of PDGF-AB was 52.76±7.89 ng/ml. The proliferation of rat and human osteoblast were promoted after treated with rat-SRGFand human-SRGF, respectively. In rat osteoblast groups, there were significant differences in absorbency between ratSPGF group and rat-SRGF group at 48 and 96 hours(Plt;0.05). In human osteoblast groups, the differences between human-SPGF group and human-SRGF group were significant at 48, 72 and 96 hours(Plt;0.05). The proliferation of these two kinds of osteoblasts almost stopped in serum-free medium, and the differences in absorbency , compared with othergroups，were significant (Plt;0.05). Conclusion High quality of PRP can be achieved by the improved method and SRGF is capable of up-regulating the proliferation of rat osteoblast and human osteoblast.
ObjectiveTo explore the effect and mechanism of netrin-1 on blood-retinal barrier permeability in diabetes mellitus (DM) rats. MethodsEighty Sprague-Dawley rats were randomly divided into the normal control group, DM+balanced salt solution (BSS) group, DM+netrin-1 low dose group and DM+netrin-1 high dose group, with 20 rats in each group. DM rats were induced by intraperitoneal injection of streptozocin (STZ). These rats were feed with high sugar and fat for 3 months after STZ injection. All rats were sacrificed at 1 month after intravitreal injection. Retinal vascular permeability was measured by Evans blue. The expression level of occludin was determined by immunohistochemistry. Hematoxylin-eosin (HE) staining of retina was used to observe the pathological change of DM and the level of occludin mRNA was analyzed by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR). Five rats of each group. ResultsHE staining of retina showed that the degree of edema and vascularization in DM+netrin-1 high dose group was better than DM+BSS group. Staining of occludin in retina was limited to nerve fiber layer, ganglion cells, inner plexiform layer and inner nuclear layer in normal rats, but in DM+BSS group, the color of staining positive of occludin was lighter and more reduced. However, DM+ netrin-1 group occludin staining was deepen and enlarged. The result of RT-PCR showed that the expression of occludin mRNA in other three groups was less than normal control group (P < 0.05). The significant difference during DM+BSS group, low dose group and DM+netrin-1 high dose group (F=177.13, P=0.00), and the more concentrate of netrin-1 the higher expression of occluding. Compared the DM+netrin-1 low dose group with DM+BSS group, there was significant difference expression of occludin (t=-13.98, P=0.00). There was significant difference between the DM+netrin-1 high dose group and normal control group (t=12.87, P=0.00). There was statistically significant difference in DM+BSS group, DM+netrin-1 low dose group and DM+netrin-1 high dose group (F=179.69, P=0.00). Compared the two group of different concentration netrin-1, the quantification of vascular permeability in DM+netrin-1 high dose group reduced more (t=12.73, P=0.00). ConclusionsNetrin-1 can protect the blood-retinal barrier in DM rats. Netrin-1 may decrease BRB leakage in DM rats by protecting the expression of occludin.
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)