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.
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.
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 regulation of PTB-associated splicing factor (PSF) exerts on phosphatidylinositol 3 kinase (PI3K)/Akt signaling pathway in cultured retinal pigment epithelial (RPE) cells. MethodsARPE-19 RPE cells were divided into five groups including PSF overexpression (0.25, 0.50, 1.00 μg of pEGFP-C2-PSF plasmid DNA), PSF overexpression control (pEGFP-C2 empty vector DNA), PSF inhibition (0.25, 0.50, 1.00 μg of pGenesil-PSF-RNAi plasmid DNA), PSF inhibition control (pGenesil-scramble-siRNA empty vector) and sham transfected group (treated with lipofactamine 2000 reagent, but without adding plasmid DNA) groups. After transfecting with plasmid DNA, the cells were stimulated with insulin-like growth factor-1 (IGF-1). IGF-1-stimulated ARPE-19 cells were also treated with Wortmannin and /or PSF over-expression. WST-1 assay was used to detect the proliferation rates, the VEGF mRNA levels were analyzed using real time polymerase chain reaction (PCR), the levels of phosphorylation Akt and total Akt expression were measured by western blotting. ResultsAfter IGF-1 stimulation, the difference of the cell proliferation rates between PSF overexpression group, PSF overexpression control group and sham transfected group was statistically significant (F=29.728, P<0.05). The difference of the cell proliferation rates between PSF inhibition group, PSF inhibition control group and sham transfected was also statistically significant (F=14.121, P<0.05). Compared with control group, the VEGF mRNA levels was decreased in PSF overexpression group (P=0.000 3), but increased in PSF low expression group (P=0.030 9). Furthermore, overexpression of PSF could down-regulate the activation of pAkt after IGF-1 stimulation. When combined with Wortmannin treatment, the VEGF mRNA levels in PSF overexpression group was significantly lower than the control group (P<0.05). ConclusionsAfter IGF-1 treatment, PSF plays a role in suppressing the proliferation and VEGF expression in RPE cells by inactivating PI3K/Akt signaling pathway.
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.
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.
Based on the pathogenic mechanisms of age-related macular degeneration (AMD), tremendous preclinical and clinical trials have demonstrated that cell transplantation which aim to replace impaired retinal pigment epithelium (RPE) with healthy RPE cells is a promising approach to treat AMD. So far, choices of cell sources mainly are autologous RPE, iris pigment epithelium, fetal RPE, human embryonic stem cell-derived RPE and human induced pluripotent stem cell-derived RPE, and some of them are undergoing clinical researches. Grafting manners in cell-based therapies are various including RPE sheet or RPE-choroid complex transplantation, RPE cell suspension injection, and RPE sheet transplantation with scaffolds. This review is limited to cell-based therapies for RPE that damaged first in the progress of AMD and focus on recent advances in cell sources, transplantation methods, preclinical and clinical trials, and the obstacles that must be overcome.
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.
In thiis study,we show thai carbachol stimulates the accumulation of inositol phosphates(InsPs)in human rellnal pigment epithelium (RPE)cells and atropine blocks the carbachol-induced effect ,suggesting the existence of musearinie acelyleholine receptors in human RPE cells. In contrast,noradrenaline,serotonin, cpidermal growth factor (EGF),isoproterenol,and NECA (5'-[N-ethyl]-carboxamido-adenosine)do not influence the basal levels of InsPs.Moreover,isoprmerenol and NECA do not affect the carhaehol elevated levels of InsPs.EGF,howcvcr,does potentiate the carhaehol stimulated elevation of InsPs in a dose-dependent manner ,suggesting an interaction between EGF and musearinie receptors in cultured human RPE cells. （Chin J Ocul Fundus Dis,1994,10:220-222）
Objective To observe the effect of polypyramidine tract binding protein-associated splicing factor (PSF) on hydrogen peroxide (H2O2) induced apoptosis of retinal pigment epithelial (RPE) cells in vitro. Methods RPE cells were cultured and divided into a normal group, normal+H2O2 group, Vec+H2O2group, PSF+H2O2 group according to the experimental design. Overexpression of PSF in RPE cells were achieved by pEGFP-PSF plasmid transient transfection into RPE cells, then RPE cells were exposed to H2O2. The morphological changes were observed by hematoxylin-eosin (HE) staining and Live/Dead staining while the survival rate of cells was detected by MTT assay. The effect of PSF on H2O2-induced RPE apoptosis was analyzed by Cell Death Detection ELISA kit. Meanwhile, intracellular reactive oxygen species (ROS) level was detected by using DCFH-DA method. Results Overexpression of PSF could effectively alleviate the morphological changes induced by H2O2 stimulation shown by HE staining, and effectively reduce dead cells number shown by Live/Dead staining. After H2O2 stimulation, the survival rate, apoptosis rate and ROS production level in PSF overexpression group were 0.68±0.12, 0.44±0.08 and 18 616±3 382.54 respectively, showing significant difference in comparison with the vector plasmid group and normal group (P＜0.05). Conclusion PSF overexpression plays a protective role in H2O2-induced apoptosis by inhibiting the production of ROS in RPE cells.