ObjectiveTo observe the protective effect of polypyrimidine bundle-binding protein-related splicing factor (PSF) over-expression on RPE cell injury induced by advanced glycation end products (AGEs).MethodsThe human RPE cells cultured in vitro were divided into three groups: normal control group (N group), blank control group (N + AGEs group), empty vector control group (Vec + AGEs group), and PSF high expression group (PSF + AGEs). group). RPE cells in N group were routinely cultured; RPE cells in N + AGEs group were only transfected but did not introduce any exogenous genes combined with AGEs induction; Vec +AGEs group and PSF + AGEs group were transfected with pcDNA The empty vector or pcDNA-PSF eukaryotic expression plasmid was introduced into RPE cells and induced by AGEs. Except the N group, the other 3 groups of cells were transfected accordingly, and were stimulated with 150 μg/ml AGEs for 72 h after 24 h. HE staining and Hoechst 33258 staining were used to observe the effect of high PSF expression on the morphological changes of RPE cells; ROS level detection was used to analyze the effect of PSF high expression on the ROS expression of RPE cells induced by AGEs; MTT colorimetric method was used to detect the high PSF expression Effects on the viability of RPE cells; Western blot was used to detect the effects of different time and dose of PSF on the expression of heme oxygenase 1 (HO-1).ResultsHE staining and Hoechst 33258 staining observation showed that the cells in group N were full in shape, the nucleus was round, the cytoplasm was rich, and the staining was uniform; the cells in N + AGEs group and Vec + AGEs group were reduced in size, the eosinophilic staining was enhanced, and the nucleus was densely densely stained. Pyrolysis and even fragmentation; the morphology of cells in the PSF + AGEs group was still full, the cytoplasm staining was more uniform, and the nucleus staining was uniform. The results of MTT colorimetry showed that high expression of PSF can effectively improve the viability of RPE cells, but this effect can be effectively antagonized by ZnPP, and the difference is statistically significant (F=33.26, P＜0.05). DCFH-DA test results showed that compared with the N + AGEs group and Vec + AGEs group, the ROS production in PSF + AGEs group decreased, the difference was statistically significant (F=11.94, P＜0.05). Western blot analysis showed that PSF protein up-regulated HO-1 expression in a time- and dose-dependent manner. The relative expression level of HO-1 at 24, 48, and 72 h after PSF protein was significantly higher than that at 0 h, and the difference was statistically significant (F=164.91, P＜0.05). The relative expression level of HO-1 under the action of 0.1, 0.5, 1.0, 1.5, and 2.0 μg PSF protein was significantly higher than 0.0 μg, and the difference was statistically significant (F=104.82, P＜0.05).ConclusionPSF may inhibit the production of ROS by up-regulating the expression of HO-1, thus protecting the RPE cells induced by AGEs.
ObjectiveTo observe the macular choroidal and retinal pigment epithelium (RPE) thickness in tilted disc syndrome (TDS). MethodsThis is a descriptive study. Thirty eyes of 22 TDS patients (TDS group) and 30 eyes of 15 normal subjects (control group) were analyzed. Among TDS group, there were 8 males (11 eyes) and 14 females (19 eyes), the average age was (9.00±2.78) years old. The best corrected visual acuity (BCVA) was 0.3-1.0, and the average spherical equivalent degree was (-3.44±2.22) DS. Among the control group, there were 8 males (16 eyes) and 7 females (14 eyes), the average age was (9.33±1.11) years old. The best corrected visual acuity (BCVA)≥1.0, and the average spherical equivalent degree was (-3.18±1.13)DS. The difference of the spherical equivalent degree between two groups was not statistically significant (t=-1.648, P=0.110). Enhanced depth imaging techniques of frequency-domain optical coherence tomography was used to measure the thickness of choroid and RPE at totally 17 sites. There sites included subfoveal, 4 sites each (500, 1000, 1500 and 2000 μm from the fovea) at the horizontal (nasal/temple) and vertical (superior/inferior) directions. ResultsThe subfoveal choroidal thickness was (235.53±51.77) μm and (273.45±60.3) μm in TDS patients and control respectively, the difference was significant(t=-2.612,P=0.011). The difference of the choroidal thickness of the other 8 horizontal sites (F=24.180) and 8 vertical sites (F=23.390) in TDS group was statistically significant (P=0.000). The TDS choroidal thickness of all horizontal sites except nasal 1000 μm site was thinner than corresponding sites of the control group (P<0.05). The TDS choroidal thickness of the subfoveal site and 4 inferior vertical sites was thinner than corresponding sites of the control group (P<0.05). The subfoveal RPE thickness was (32.56±5.00) μm and (36.58±3.60) μm in TDS patients and control respectively, the difference was significant(t=-3.567,P=0.001). The subfoveal RPE thickness was the thickest among other 16 sites in both groups, and the TDS RPE thickness of all sites was thinner than control group, the difference was statistically significant (P<0.05). ConclusionThe choroidal and RPE thickness of TDS patient was thinner than normal subjects.
Torpedo maculopathy is a rare, congenital lesion of RPE, which locates temporal to the macula and along the horizontal raphe. The lesion is torpedo-shaped with its torpedo-like tip pointing towards the fovea. As an incidental finding, it often affects only one eye with no damage to central visual acuity. According to its characteristics on OCT, it is divided into 2 types: typeⅠ, attenuation of outer retinal structures without outer retinal cavitation; typeⅡ, those with both attenuation of outer retinal structures and outer retinal cavitation. Diseases with pigment changes in the RPE layer similar to torpedo maculopathy include congenital hypertrophy of the RPE, RPE lesions in Gardner syndrome, etc. The main point to distinguish the disease from other diseases is its unique location and shape. Most of the torpedo maculopathy lesions are stable and do not require special treatment, but the disease can be complicated by neurosensory retinal detachment, choroidal neovascularization and so on, and symptomatic treatment is needed if necessary.
ObjectiveTo evaluate the functional and anatomical outcomes of autologous single retinal pigment epithelium (RPE) transplantation for severe obsolete submacular hemorrhage (SMH) in late age-related macular degeneration (AMD). MethodsA retrospective clinical study. From January 2012 to December 2015, 11 patients with AMD (11 eyes) with obsolete SMH who were diagnosed and treated by pars plana vitrectomy (PPV) combined with autologous RPE transplantation at the Department of Ophthalmology, Xin Hua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine were included. Among them, there were 9 eyes in 9 males and 2 eyes in 2 females. All the eyes underwent the examinations of best corrected visual acuity (BCVA) and optical coherence tomography; 4 eyes underwent macular fixation function (MAIA) at the same time. The BCVA examination was carried out using the international standard visual acuity chart, which was converted into logarithm of the minimum angle of resolution (logMAR) visual acuity during statistics. All eyes were treated with PPV combined with autologous single-layer RPE transplantation or autologous RPE-choroidal full-thickness transplantation, and were divided into S group and C group, with 5 and 6 eyes respectively. The differences of age (t=－0.363), gender composition ratio (χ2=0.549), course and thickness of SMH (t=0.118, 0.231), average times of anti-vascular endothelial growth factor drug treatments (t=0.129), times of PPV (t=－0.452) between the two groups were not statistically significant (P>0.05). The follow-up period was 6-40 months after the operation, and the BCVA, MAIA, graft status and complications of the eyes after the operation were observed. The comparison of continuous variables between groups was performed by independent-sample t test; the comparison of categorical variables was performed by χ2 test. ResultsAt the last follow-up, the average logMAR BCVA of the eyes in group S and C were 1.62±0.34 and 1.03±0.20, respectively; group C was better than group S, however, the difference was not statistically significant (t=1.532, P=0.160). There were 4 eyes (80%, 4/5) and 6 eyes (100%, 6/6) in S group and C group with BCVA better than preoperative, the difference was no statistical significance (χ2=0.677, P=0.895). There were 2 (40%, 2/5) and 3 (50%, 3/6) eyes with logMAR BCVA better than 1.0 in S group and C group, and the difference was not statistically significant (χ2=0.572, P=0.423). After the operation, 6 eyes of grafts were in good condition and 5 eyes were in poor condition; the BCVA of grafts in good condition was significantly higher than that of poor condition, the difference was statistically significant (t=4.894, P=0.001). Among the 4 eyes that underwent MAIA examination, 2 eyes were unstable and diffusely fixed on the graft; the fixation point was located at the normal retina adjacent to the graft area in 2 eyes. Secondary subretinal hemorrhage occurred in 3 eyes after the operation; the intraocular pressure was high in 1 eye after the operation. During the follow-up period, no intraocular infection, secondary retinal detachment, recurrent choroidal neovascularization or low intraocular pressure occurred in all eyes. ConclusionsBoth autologous single-layer RPE transplantation and autologous RPE-choroidal full-thickness transplantation can help stabilize or even improve the visual function of eyes with severe SMH secondary to advanced AMD. The visual acuity after surgery is closely related to the state of the graft.
ObjectiveTo investigate the role of sonic hedgehog (Shh) signal transduction pathway in the expression of vascular endothelial growth factor (VEGF) under hypoxia in cultured human retinal pigment epithelial (hRPE) cells. MethodsARPE-19 were cultured and divided into normal ARPE-19 (Cont) and hypoxia group (100 μmol/L CoCl2 Cobalt Chloride +ARPE-19); hypoxia group was further divided into CoCl2 group, cyclopamine group (CYA) and dimethyl sulfoxide (DMSO) group. 20μmol/L cyclopamine was added to the CYA group 1 hour before hypoxia, 1‰DMSO was added into DMSO group at the same time. The hRPE cells were cultured under hypoxia for 4, 8, 12, 24 hours. The expression of Shh and VEGF were determined by Real-time fluorescent quantitate PCR (RT-PCR). The amount of VEGF in the hRPE-conditioned supernatant was measured using enzyme linked immunosorbent assay (ELISA) at 4, 8, 12, 24 hours, respectively. ResultsRT-PCR tests showed that the level of Shh and VEGF of hRPE was time dependently increased (Shh: F=45.260, P=0.001; VEGF: F=264.938, P=0.001). The level of Shh and VEGF of hRPE in the group treated with cyclopamine was decreased (P < 0.01). ELISA tests showed that the amount of VEGF in hRPE supernatant was significantly increased in time-dependent manner (F=3 156.676, P=0.001), and it was down-regulated by cyclopamine under hypoxia (P < 0.01). ConclusionShh signal transduction pathway could play a role in the VEGF expression induced by hypoxia in hRPE cells.
Objective To investigate the protective effects of riluzole, a sustained activator of K2P subfamily member TRAAK potassium channel, in human retinal pigment epithelium (hRPE) cells with oxidative induce by tert-butyl hydroperoxide (t-BHP) in vitro, and to evaluate the possible involvement of K2P in the cytoprotective function of retina degeneration diseases. Methods The third to fifth passage of the primary cultured hRPE cells were used in the following experiments．hRPE cells were divided into seven groups: normal control group.t-BHP (300 mu;mol/L) group.t-BHP with riluzole (2, 5, 10, 20 mu;mol/L) group and riluzole (10 mu;mol/L) group. The apoptosis was measured by the 3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, annexinV/PI double staining flow cytometry. Changes of cells and nuclei morphology were observed under a phase contrast microscope and a fluorescence microscope after 4prime;, 6-diamidino-2-phenylindole (DAPI) staining. Immunofluorescence 1abelling was carried out to analysis the expression of TRAAK. Results After 24 hours incubation with 300 mu;mol/L t-BHP, the cells viability decreased to (58.7plusmn;12.2)% as compared to the normal control groups. The cell viability of t-BHP with riluzole group at different concentrations was higher than the t-BHP group, while 10 mu;mol/L riluzole showed maximally protective effect on hRPE death induced by t-BHP（t=4.84.P＜0.05）. Riluzole remarkably decreased pyknotic nucleus and cell swelling when compared with t-BHP group. Morphology of cells was fusiform with the uniform elliptic nuclei in normal and riluzole group. The Results of annexinV/PI double staining flow cytometry showed that ratio of normal cells were （97.6plusmn;1.3）%, （70.3plusmn;7.0）%, （86.9plusmn;5.2）%, （93.9plusmn;1.5）% in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. The ratio significant decreased in t-BHP group when it was compared with the other groups (t=7.53, 4.59, 6.49, respectively.P＜0.05). By contrast with normal group and riluzole group, the ratio of normal cells in t-BHP with riluzole group had no statistical significance（t=2.94, 1.91, respectively.P＞0.05）. Riluzole (10 mu;mol/L) also significantly decreased the ratio of early stage apoptotic cells from (25.50plusmn;8.02)% to (1.20plusmn;0.72)% in t-BHP injured groups （t=7.13，P＜0.05）. The ratio of early stage apoptotic cells significant decreased in t-BHP group when it was compared with the normal group and riluzole group （t=7.07, 5.94, respectively.P＜0.05）. By comparison with normal group and riluzole group, there are no statistical significance in t-BHP with riluzole group（t=0.06, 1.18, respectively.P＞0.05）. The mean gray values of TRAAK expression were 0.040plusmn;0.003, 0.041plusmn;0.001, 0.049plusmn;0.001, 0.055plusmn;0.001 in normal group.t-BHP group.t-BHP with riluzole group and riluzole group respectively. TRAAK density was significantly higher in t-BHP with riluzole group and riluzole group（t=7.40, 12.70, respectively.P＜0.05）. Conclusions Riluzole can protect hRPE cells against oxidative injury-induced cell death at early apoptosis stage. The mechanism may relate to that riluzole can promote the expression of K2P TRAAK potassium channel.
ObjectiveTo observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress. MethodsThe third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier. The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10% normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro. hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group. Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours. Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment. TER was measured to evaluate the barrier function of hRPE. The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2), together with complement bioactive fragments (C3a, C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay. ResultsStable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert. Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51% compared with normal hRPE barrier. CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48% compared with normal hRPE barrier(t=21.60, P < 0.05). Compared with model group, CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48% and 23.47% secreted by hRPE under complement-activated oxidative stress (t=3.26, 2.43; P < 0.05). Compared with model group, CR1 treatment could also decreased the concentration of C3a, C5a and MAC by 24.00%, 27.87%, 22.44%.The difference were statistically significant (t=9.86, 2.63, 6.94; P < 0.05). ConclusionsCR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress. The underlying mechanism may involve inhibiting complement activation and down-regulating the expression of VEGF and CCL2.
ObjectiveTo observe the effect of exosomes derived from human umbilical cord blood mesenchymal stem cells (hUCMSC) on the expression of vascular endothelial growth factor (VEGF) A in blue light injured human retinal pigment epithelial (RPE) cells. MethodshUCMSC were cultured with exo-free fetal bovine serum for 48 hours, and then the supernatants were collected to isolate and purify exosomes by gradient ultracentrifugation method. Transmission electron microscopy was used to identify the morphology of exosomes. Surface specific maker protein CD63 and CD90 were detected via Western blot. Cultured ARPE-19 cells were divided into normal control group, blue light injured group and hUCMSC exosomes treated group. Cells were exposed to the blue light at the intensity of (2000±500) Lux for 12 hours to establish the light injured models. The cells of hUCMSC exosomes treated group were treated by different concentrations of exosomes for 8, 16, 24 hours. The mRNA and protein of VEGF-A were determined by real time-polymerase chain reaction and Western blot. Immunofluorescence assay were used to detect the expression levels of VEGF-A. ResultshUCMSC exosomes were successfully isolated, they exhibited round or oval shape and their diameter ranged from 50 to 100 nm with membrane structure through electron microscope. hUCMSC exosomes expressed the common surface marker protein CD63 and the surface marker protein CD90 of hUCMSC. The protein and mRNA level of VEGF A in the blue light injured group increased significantly compared to that in normal control group (t=-16.553, -19.456; P < 0.05). After treating with low, middle and high concentration of hUCMSC exosomes for 8, 16 and 24 hours, the protein and mRNA level of VEGF A of injured RPE were significantly decreased (P < 0.05). With the treated time and concentration of hUCMSC exosomes improved, the protein and mRNA level of VEGF A of injured RPE gradually decreased (P < 0.05). Immunofluorescence assay showed the protein level of VEGF-A of injured RPE gradually decreased with the same concentration of hUCMSC exosomes treated over time. ConclusionhUCMSC exosomes can effectively down-regulate the mRNA and protein level of VEGF-A in blue light injured RPE, the effect depends on the concentration and treated time of hUCMSC exosomes.
RCBTB1 gene associated hereditary retinopathy is an extremely rare inherited retinal disease (IRD) discovered recently. The mutation of RCBTB1 gene can lead to a variety of IRD clinical phenotypes, such as early retinitis pigmentosa and delayed chorioretinal atrophy. The hereditary mode of RCBTB1 gene associated retinopathy is autosomal recessive. RCBTB1 gene plays an important role in maintaining mitochondrial function and anti-oxidative stress defense mechanism of retinal pigment epithelium cells. In the future, it is necessary to further determine whether there is a genotypic and phenotypic correlation in the age of onset of RCBTB1 gene associated retinopathy or multi-organ involvement, and evaluate the safety and efficacy of adeno-associated virus-mediated RCBTB1 gene replacement therapy in animal models, to explore the feasibility of gene replacement therapy and stem cell therapy.
Severely coagulated retinae by argon laser of 20 Chinese hamsters were investigated with transmission electron-microscopy. The results revealed destruction of retinal pigment epithelium-Bruch's membrane-choroid capillary complex at the coagulated foci, and leakage of fluid and blood cells through the choroidal vessels into the subretinal space. Several days after laser burn the subretinal fluid was found to subside and the RPE cells surrounding the burned lesions started to proliferate. The smaller lesions were covered by the proliferating RPE 10 days after coagulation, but poor regeneration of RPE in large necrotic areas. Neovascularization was usually associated with obvious defect of Bruch's membrane and restoration of RPE barrier was most likely impossible. （Chin J Ocul Fundus Dis,1992,8:14-16）