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.
ObjectiveTo observe the effect of TGF-β receptor inhibitor Compound C on the directed differentiation of human embryonic stem cells (hESC) into retinal pigment epithelial (RPE) cells.
MethodsH1 hESC were divided into control group and experimental group. When the hESC reached over confluence, the medium was changed to knockout serum replacement medium without bFGF to induce RPE differentiation. The experimental group was supplemented with 1 μmol/L TGF-β receptor inhibitor Compound C at the first six days of induction. Real-time PCR was carried out to examine the expression of paired-box gene 6 (PAX6), microphthalmia-associated transcription factor (MITF), cellular retinaldehyde blinding protein (CRALBP), and RPE65 in both groups at the 1, 3, 5 weeks of the induction process. hESC-derived RPE (hESC-RPE) cells were isolated mechanically and purified. Real-time PCR, Western blot and immunofluorescence were used to characterize the purified hESC-RPE cells.
ResultsPigmented colonies were observed in experimental group at the 4 weeks of the induction process, while no pigmented colony could be detected in the control group. All the purified pigmented cells from experimental group showed polygons morphology. Experimental group showed significantly higher expression of RPE marker genes PAX6, MITF, CRALBP and RPE65 than the control group(P<0.05). Compared with the hESC and ARPE-19 cells line, purified hESC-RPE cells showed much higher expression of PAX6, MITF, CRALBP and RPE65(P<0.05).High expression level of PAX6 and RPE65 proteins were observed in hESC-RPE cells. Immunofluorescence verified the expression of PAX6 and ZO-1 in hESC-RPE cells.
ConclusionTGF-β receptor inhibitor Compound C significantly improved the differentiation efficiency of hESC into RPE.
Objective To investigate the effects of exosomes from cultured human retinal pigment epithelium (ARPE-19) cells affected by oxidative stress on the proliferation and expression of vascular endothelial growth factor-A (VEGF-A) and Akt of ARPE-19 cells. Methods Culture ARPE-19 cells. The concentration of 2.5 μmol/L rotenone was selected to simulate oxidative stress and isolated ARPE-19-exosome. Exosomes were isolated by ExoQuick exosome precipitation solution. Transmission electron microscopy was used to identify the morphology of exosomes. Western blot was used to detect exosomes’ surface-specific maker protein CD63. ARPE-19 cells affected by oxidative stress were cultured with exosome as experimental group, normal ARPE-19 cells were cultured with exosome as control group. The cell proliferation was examined by methyl thiazolyl tetrazolium assay. Western blot and immunofluorescence assay were used to detect the expression levels of VEGF-A and Akt protein. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the levels of VEGF-A mRNA and Akt mRNA. Results The diameter of normal ARPE-19-exosomes ranged from 50 to 150 nm. The isolated exosomes expressed CD63. AREP-19 cells were cultured with ARPE-19 (affected by rotenone)-exosome, the cell viability in experimental group was significantly reduced than in the control group. Green fluorescence was observed in the cytoplasm under fluorescence microscope. Compared with the control group, VEGF-A was up-regulated expressed and Akt was down-regulated expressed. Western blot results showed that, VEGF-A protein expression in the experimental group were higher than the control group. Akt protein expression in the experimental group were less than the control group. The difference was statically significant (t=3.822, 6.527;P＜0.05). RT-PCR results showed that VEGF-A mRNA expression levels was higher in the experimental group than the control group. Akt mRNA expression levels was lower in the experimental group than the control group. The difference was statically significant (t=8.805, ?7.823;P＜0.05). Conclusions Exosomes from ARPE-19 cells affected by oxidative stress inhibit the proliferation of normal ARPE-19 cells, increase the expression of VEGF-A and reduce the expression of Akt.
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.
Objective
To investigate the impact of photodynamic therapy (PDT) with verteporfin on the expression of pigment epithelial derivative factor (PEDF) mRNA and vascular endothelial growth factor (VEGF) mRNA in adult retinal pigment epithelial (RPE) cells in vitro.
Methods
The changes of cellular viability before and after PDT were assessed by methyl thiazolyl tetrazolum (MTT) colorimetric assay. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) was conducted to detect the expression of PEDF and VEGF mRNA in RPE cells before and after PDT.
Results
PDT caused the death of RPE cells. The cellular mortality was positively correlated with the power of photocoagulation and the concentration of verteporfin.
Conclusion
PDT could downregulate the expression of PEDF and VEGF mRNA in adult RPE cells in vitro, which may relate to the cure or relapse of subfoveal choroidal neovascular membrane after PDT.
(Chin J Ocul Fundus Dis, 2006, 22: 256-260)
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.
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.
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）
Retinitis pigmentosa (RP) is a genetic disorder of photoreceptor cell apoptosis and retinal pigment epithelium (RPE) cell atrophy caused by gene mutation. The clinical manifestations are night blindness, peripheral visual field loss and progressive vision loss. RPE cell apoptosis plays an important role in the progression of RP, and exogenous implantation of RPE cells as an alternative therapy has shown certain efficacy in animal experiments and clinical trials. With the diversification of cell sources, the update of surgical techniques and the continuous emergence of biological materials, more possibilities and hopes are provided for cell therapy. To further promote the development of this field in the future, it is still necessary to strengthen the cooperation between medicine, bioengineering and other disciplines in the future to jointly promote the innovation and development of therapeutic methods. It is believed that RPE cell transplantation therapy will show a brighter prospect in the future
ObjectiveTo study the role of Rac1 in the epithelial-mesenchymal transition (EMT) process of retinal pigment epithelial cells (RPE) induced by transforming growth factorβ(TGF-β).
MethodsHuman ARPE-19 cells were divided into 4 groups including control group, TGF-βgroup, TGF-β+NSC23766 group, NSC23766 group. NSC23766 was added to medium 2 hours before TGF-βtreatment to block the Rac1 receptors.α-smooth muscle actin (α-SMA) expression was measured by immunofluorescence and Western blot. Cell scratch assay, invasion assay and gel contraction experiments were used to measure cell migration, invasion, cell contraction.
ResultsThe expression ofα-SM A was higher in TGF-βgroup, compared with the control group, TGF-β+NSC23766 group (F=825.314, P < 0.05). Cell scratch assay showed that the cellular gap was less in GF-βgroup, compared with the control group, TGF-β+NSC23766 group, NSC23766 group (F=177.351, P < 0.05). Cell invasion assay showed that, the number of cells pass through the fiber membrane was the same in TGF-βgroup and other 3 groups (F=0.371, P=0.055). Gel contraction assay showed that TGF-βcan promote the cellular contraction, compare to the control group, TGF-β+NSC23766 group, NSC23766 group, the difference was statistically significant (F=40.473, P < 0.05).
ConclusionRac1 play a role in TGF-β-induced behavioral changes of RPE cells; NSC23766 inhibit RPE cellular behavior change by regulating Rac1 activation.
