ObjectiveTo investigate the effects of leptin on the oxidative damage in human retinal pigment epithelial (RPE) cells. MethodsHuman RPE cells (ARPE-19) were cultured in vitro, and randomly divided into control group and insulin resistance group. RPE cells were treated with 0, 10, 100 ng/mL leptin for 24, 48, 72 hours respectively. Then the levels of reactive oxygen species (ROS) expression in RPE cells were detected by 2', 7'-dichlorofluorescin-diacetate (DCFH-DA), and the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in RPE cells were observed by immunocytochemistry (ICC), and the levels of human 8-oxoguanine DNA glycosylase l (hOGG1) expression in lysate were measured by Western blot. ResultsAfter 24, 48, 72 hours, the level of ROS (Control group:F=37.136, 37.178, 49.634; P < 0.05. Insulin resistance group:F=9.822, 28.881, 71.150;P < 0.05), 8-OHdG (Control group:F=88.643, 390.920, 1039.276;P < 0.05.Insulin resistance group:F=273.311, 299.155, 82.237;P < 0.05) and hOGGl (Control group:F=470.062, 1073.113, 295.456;P < 0.05. Insulin resistance group:F=240.032, 592.389, 527.760;P < 0.05) expression increased significantly with the increase of leptin concentration in control group and insulin resistance group. Under the same leptin concentration, the level of 8-OHdG has a trend that it was higher in the insulin resistance group than the control group. After 24 hours, the difference of hOGGl expression between control group and insulin resistance group was not significant (F=23.392, P > 0.05). After 72 hours, the level of hOGGl expression was significantly higher in the insulin resistance group than the control group (F=129.394, P < 0.05). The level of hOGGl expression was significantly higher at 48 hours than that at 24 hours and 72 hours (P < 0.05). ConclusionLeptin could induce the oxidative damage of RPE cells in normal and insulin resistance status. With the increase of leptin concentration and time extended, the degree of oxidative damage and its repair were both increased. The degree of oxidative repair increased with the increase of leptin concentration, but decreased with time extended.
ObjectiveTo investigate the impact of L-Phenylalanine on the efficiency of retinal pigment epithelial (RPE) cell derivation from human embryonic stem cells (hESCs) and explore the underlying mechanisms. MethodsH1 hESCs were routinely cultured with mTeSR medium and divided into control and experimental groups. When cells reached over-confluence, spontaneous differentiation was triggered using 10% KSR differentiation medium without bFGF. L-Phenylalanine (0.2 mmol/L) was supplemented in the experimental group from the 3rd week. The expression of RPE markers and Wnt signaling components in the two groups was detected by Real time-RCR, Western blot and Flow cytometry analyses. Purified hESC-RPE cells and PBS were injected into the subretinal space of sodium iodine-induced retinal degeneration rats separately. Retinal function was assessed by ERG 6 weeks after the transplantation. ResultsOn the 7th week, much more pigment cell clumps appeared in the experimental group compared to the control group. Within these areas there were monolayer hexagonal RPE cells full of pigment granules. The experimental group showed significantly higher expression of Pax6, MITF, Tyrosinase, RPE65, Wnt3a, Lef1 and Tcf7 genes than the control group (P < 0.01). Higher expression level of MITF and RPE65 proteins and higher percentage of RPE65 (+) cells (P < 0.01) were detected in the experimental group. 6 weeks after sub-retinal transplantation of hESC-RPE cells, the amplitudes of a-b wave in the transplanted eyes were significantly higher than those in the control eyes (P < 0.01) at the stimulus intensity of 3.0 cd·s/m2. ConclusionsL-Phenylalanine effectively promoted the differentiation of embryonic stem cells into retinal pigment epithelial cells, and its impacts on the Wnt/β-catenin signaling pathway may partially explain the underlying mechanisms. Subretinal transplantation of hESC-RPE remarkably improved the retinal functions of retinal degenerative animal models.
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）
ObjectiveTo observe the effect of subretinal injection of retinal pigment epithelium (RPE) cells for RPE in mice. MethodsA total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group, OIR model group and OIR model cell transplanted group, 10 mice in each group. The OIR model was induced in mice of OIR model group and OIR model cell transplanted group. The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group. At 20 days after the injection, the RPE thickness was evaluated by fluorescence microscope. The expression of RPE65, Bestrophin and zonula occludens-1 (ZO-1) were estimated by Western blot and real-time quantitative PCR (RT-PCR). ResultsThe thickness of RPE in OIR model mice was thinner than that in normal mice; the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice. The results of Western blot and RT-PCR indicated that the differences of protein (F=8.597, 18.864, 25.691) and mRNA expression (F=39.458, 11.461, 34.796) of RPE65, Bestrophin, ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group (P < 0.05). ConclusionsSubretinal injection of RPE cells can promote RPE thickening. RPE65 and Bestrophin protein relative expression levels increased, ZO-1 protein relative expression levels reduced; mRNA expression levels of RPE65, Bestrophin and ZO-1 genes increased.
Replacement of diseased retinal pigment epithelium (RPE) cells with healthy RPE cells by transplantation is one option to treat several retinal degenerative diseases including age-related macular degeneration, which are caused by RPE loss and dysfunction. A cellular scaffold as a carrier for transplanted cells, may hold immense promise for facilitating cell migration and promoting the integration of RPE cells into the host environment. Scaffolds can be prepared from a variety of natural and synthetic materials. Strategies, such as surface modification and structure adjustment, can improve the biomimetic properties of the scaffolds, optimize cell attachment and cellular function following transplantation and lay a foundation of clinical application in the future.
Objective To investigate the expression of eotaxin-1, eotaxin-2 and eotaxin-3 in ARPE-19 human RPE cells after exposure to light. Methods Cultured human RPE cells (5th~10th generations) were divided into lightinduced group and control group. Cells light-induced group were exposed to the blue light at the intensity of (600plusmn;100) Lux for 12 h to establish the light damaged model. Eotaxin-1, eotaxin-2 and eotaxin-3 mRNA and protein were determined by real time polymerase chain reaction and Western blot at 0, 3, 6, 12, 24 hours after light-induced. Results In light-induced groups, mRNA levels of eotaxin-1 and eotaxin-2 were increased at 0 h (t1=6.05.t2=12.561) and 3 h （t1=2.95.t2=3.67） significantly(P＜0.05), but the mRNA level of eotaxin-3 had not changed （t3=1.57 and 1.00 respectively，P＞0.05） at that time. At 6 h （t1=4.73，t2=18.64，t3=28.48）, 12 h （t1=3.11，t2=20.62，t3=18.50）, 24 h （t1=8.25，t2=38.27，t3=18.60）, mRNA levels of eotaxin-1, 2, 3 were increased significantly （P＜0.05）. Except for the eotaxin-3 protein had not changed at 3 h （t3=1.28，P＞0.05）, protein expression of eotaxin-1, 2, 3 were increased significantly （P＜0.05） at 0 h （t1=4.85，t2=5.45，t3=6..21）, 3 h （t1=5.64，t2=4.55）, 6 h （t1=31.60，t2=6.63，t3=7.15）, 12 h （t1=14.09，t2=18.22，t3=15.76）, 24 h （t1=6.96，t2=10.47，t3=12.85）. Conclusion Eotaxin-1, eotaxin-2 and eotaxin-3 expression were increased after Light-damage, corresponding to the time after light exposure. Eotaxin-3 was the most prominent isoform.
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 changes of eotaxin-1(CCL11), eotaxin-2(CCL24)and eotaxin-3(CCL26)in ranibizumab treated light-injured human retinal pigment epithelium (RPE) cells ARPE-19 and investigate the effects of vascular endothelial growth factor (VEGF) antagonist to the expressions of eotaxins. MethodsCultured human RPE cells(8th-12th generations)were divided into light-injured group, ranibizumab treated group and normal control group. Cells of the three groups were exposed to the blue light at the intensity of(600±100) Lux for 12 h to establish the light injured model, while cell culture dishes of the normal control group were wrapped with double-layer foil. The cells of ranibizumab treated group were treated with VEGF-A antagonist(ranibizumab)at the final concentration of 0.125 mg/ml for 24 hours directly after the illumination. The mRNA and protein of VEGF-A, eotaxin-1, eotaxin-2, eotaxin-3, NF-κB were determined by Real time-PCR, enzyme-linked immunosorbent assay, Western blot, immunohistochemical staining at 0, 3, 6, 12, 24 hours after light damage. ResultsThe mRNA and protein level of VEGF-A, eotaxin-1, eotaxin-2, eotaxin-3, NF-κB in the light-injuried group increased significantly compared to that in normal control group (P < 0.05). After treating with ranibizumab, the expression of eotaxin-1, eotaxin-2, eotaxin-3, NF-κB were significantly suppressed (P < 0.05). ConclusionThe suppression of over-expression of VEGF in human RPE may down-regulate the expression of eotaxins, via the suppression of NF-κB.
Objective To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells. MethodsSARS-CoV-2 S-protein gene fragment expression plasmid (p3xflag-S) was constructed and transfected into human RPE, HEK293 cells. DNA sequencing was used for identification, and the expression of Flag-S was detected by Western blot. HEK293 cells were divided into the cells 1, 2, 3 and 4 and transfected with GFP11 plasmid and vector, GFP1-10plasmid and vector, transfected with GFP11 and pCMV-HA-ACE2 plasmid, GFP1-10 and p3xflag-S plasmid. Cell 1 was co-cultured with cell 2 (control group 1), cell 2 with cell 3 (control group 2), cell 3 with cell 4 (observation group), and cell 1 mixed with cells 2, 3 and 4 (control group 3). Bright-field microscopy and fluorescence microscopy were used to observe cell fusion. RPE cells were divided into control group and overexpression S-protein group. The cell cycle was detected by flow cytometry; the cell proliferation level was detected by Counting Kit 8 (CCK-8); and the S-protein expression level in RPE cells was detected by Western blot. The Student’s t-test was performed for comparison between groups. ResultsDNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed thatS-protein-related expression was elevated in transfected HEK293 cells compared with untransfected p3xflag-S cells. Large, multinucleated fused cell clusters were visible under bright-field microscopy; multiple nuclear with distinct green fluorescence were visible in the fused cells under fluorescence microscopy. Western blot assay showed elevated S-protein-related expression in transfected p3xflag-S plasmid RPE cells compared to untransfected p3xflag-S plasmid RPE cells. CCK-8 results showed that the proliferative capacity of RPE cells in the S-protein overexpression group was significantly reduced compared with the control group, with statistically significant differences (t=22.70, 16.75, 23.38; P<0.000 1). The results of flow cytometry showed that the G1 phase cells in the control and overexpression S-protein groups were 41.1% and 67.0%, respectively; compared with the control group, the G1 phase cells in the overexpression S-protein group were significantly higher, and the difference was statistically significant (t=4.76, P=0.018). The apoptosis rate was significantly increased in the S-protein overexpression group compared with the control group, and the difference was statistically significant (t=4.91, P=0.008). ConclusionOverexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.
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.