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Background Pluripotent stem cell-derived retinal pigment epithelial (RPE) cells holds great promise for the treatment of age-related macular degeneration (AMD) and retinitis pigmentosa (RP),but the poor induction efficiency and the according high cost of RPE differentiation hindere its clinical applications.Curcumin is proved to have a promoting effect on the induced differentiation of embryonic stem cells (ESCs).However,the mechanism of curcumin on differentiation of human ESCs into RPE-like cells remains unclear.Objective This study aimed to explore the underlying molecular mechanism of curcumin on directed differentiation of human ESCs into RPE-like cells.Methods Human ESCs strains were cultured in the Matrigel-coated 6-well plate with mTeSRTM 1 medium until over-confluence,and basic fibroblast growth factor was withdrawn there after to induce automatic differentiation.Curcumin at the final concentration 1 μmol/L was added in the first day of differentiation for 24 hours,and the cells without curcumin in the medium served as the control group.Total RNA and protein were extracted at 3 weeks and 5 weeks after induction.RT-PCR,Western blot and immunofluorescence were performed to examine the expressions of the biomarks of stem cells and RPE cells as well as Wnt/β-catenin signaling pathway components.The endocytosis of polystyrene microsphere by induced RPE (iRPE) cells was investigated to verify their function of phagocytosis which features RPE cells.Results Pigmented cells were found from 3 weeks through 5 weeks after induction in the curcumin group,but only less pigmented cells were seen in the fifth week after induction in the control group.In the third and fifth week after induction,the relative expression levels of NANOG mRNA in the iRPE cells were significantly lower than those in the control group (t =13.086,P =0.022;t =34.186,P =0.004),and the relative expression levels of Pax6,RX,CRALBP and RPE65 mRNA were higher in the curcumin group than those of the control group (all at P＜0.01).Western blot assay showed that the expressing bands for CRALBP,RPE65 and MITF enhanced in iRPE cells with a similar appearance in human RPE cells.However,these expressions were all absent in human ESCs.Immunofluorescence staining showed the positive expressions of Pax6,MITF and ZO-1 in cytoplasm of iRPE cells in the curcumin group with a purified efficacy 100％.The fluorescence dye-doped polystyrene microspheres in cytoplasm were obvious in the iRPE cells like positive controls,but the polystyrene microsphere was absent in the negative controls.From 3 weeks through 5 weeks after induced,the relative expression levels of Lef1,MYC and TCF7 mRNA (the dwnstream target genes of Wnt signaling pathway),FZD3 mRNA (Wnt receptor),Wnt2B mRNA (Wnt ligand) and Wnt7B mRNA were significantly reduced in the curcumin group compared with the control group (all at P＜0.01).Conclusions Curcumin promotes the differentiation of human ESCs into RPE-like cells by stimulating the activation of Wnt signaling pathway,and therefore accelerate the differentiation and mature of iRPE cells.
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ObjectiveTo detect the features of dopamine secretion of cultured bovine retinal pigmentary epithelial(RPE) cells. The level of dopamine and survival rate after passage and microencapsulation were also observed. MethodsThe primary culture of bovine RPE cells was made using enzyme digestion. After purification and identification the growth curve of the cell was observed. Alginate-polylysine-alginate(APA) microencapsulated cells were made with a high voltage electostatic system. The activity of the cells in the mirocapsule was investigated by trypan blue staining. The secretion of dopamine was determined by high performance liquid chromatography (HPLC) assay. ResultsThe cell had high purity of immunocytochemistry. The growth curve showed that the exponential growth occurred at the first 1~4 days. Dopamine content was first detected at the time point of 2 hour, and arrived at the peak at about 48 hour of the cultivation. The secretion of dopamine was not different between passages. Dopamine secretion was dramatically decreased in the first 4 days after microencapsulation (P
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Objective To observe the effect of visible light on apoptosis of cultured human retinal pigment epithelium (RPE) cells. Methods Being the light source,500lx,(2 000?500)lx and (3 400?200)lx cold white light were used. The duration of exposure was 0,6,12 and 24 hours respectively. Apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labelling, Annexin V flunorescein isothiocyanate/Propidium iodium labelling and flow cytometry. Results Apoptosis and necrosis were found in cultured human RPE cells which were exposed to visible light.(1)A significant increase in apoptotic and necrotic percentages was consistent with a higher light intensity.(2)Apoptosis was the main response to shorter (6 h and 12 h) exposure duration,while necrosis was more pronounced correlated to the prolongation of post exposure culture ( P 500 lx) increases the proportion of apoptosis and necrosis of human RPE cells in vitro.The extent is related to exposure intensity and duration. It demonstrates that the lower intensity and the shorter duration of exposure to light are, the more pronounced apoptotic percentages are observed,otherwise necrosis.
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Objective To verify whether iris pigment epithelial cells (IPECs) possess the similar potential of specific phagocytosis to retinal outer segments (ROS) with retinal pigment epithelial cells (RPECs). Methods IPECs were isolated from neonatal bovines with Hu′s method, and were cultured. The cultured cells were identified by immunohistochemical methods with antibodies to cytokeratin and S 100. Total RNA of IPECs was extracted by Trizol. The specific primers for mannose receptor and ? actin were designed according to their sequence from Genbank. The mRNA expression of these proteins in the IPECs was analyzed by reverse transcription polymerase chain reaction (RT PCR). Results The cultured IPECs have no contamination of other cells. The extracted RNA was ideal and had no degradation. RT PCR analysis showed that mannose receptor′s mRNA was expressed in cultured IPECs in vitro. Conclusions Cultured IPECs may express the mannose receptor, and may have similar potential of phagocytosis to ROS with RPECs.
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Objective To investigate the effect of subretinal fluid (SRF) with different grades of proliferative vitreoretinopathy (PVR) on bcl-2 oncoprotein expression in retinal pigment epithelium (RPE) cells and fibroblast (FB). Methods Using immunohistological staining technique and Western-blotting method to detect the expression of bcl-2 protein in RPE cells and FB under the stimulation of SRF. Results The expression levels of bcl-2 increased in both types of cells to a certain extent compared with those of the control group 4 hours after the cells subjected to SRF; 36 hours later, the expression levels of bcl-2 of experimental groups decreased more quickly than those of the control group,and the control group showed relatively higher bcl-2 protein levels at the end of observation. Conclusions SRF may stimulate the expression of bcl-2 in RPE cells and FB under culture at early stage, but accelarate the declining of bcl-2 protein levels a certain time after subjected to SRF.
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Objective To investigate the protective effect of nerve growth factor (NGF) on apoptosis of cultured human fetal retinal pigment epithelium (HFRPE) cells induced by indomethacin (IN) in vitro. Methods Subcultured HFRPE cells were treated with different concentrations of IN to establish apoptotic model. The protective effect of NGF on apoptosis of cultured HFRPE cells were assessed using an acridine orange (AO) staining method and transmission electron microscopy (TEM). Results HFRPE cells exposed by 200 600 ?mol/L IN for 24 hours elicited typical apoptosis morphological changes, including condensed chromation, nuclear fragmentation and reduction of nuclear size and cell volume. There was a statistically difference in HFRPE cells with apoptosis between 200 ?mol/L IN+500 ?g/L NGF and 200 ?mol/L IN groups ( q=3 9204,P=0.0320) ; there was a significant difference in HFRPE cells with apoptosis in 400 ?mol/L IN+500 ?g/L NGF and 400 ?mol/L IN as well ( q=9 7915,P=0 0001). Conclusion NGF has an protective effect on IN induced HFRPE cells apoptosis.
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Objective To investigate the effect of hypericin on the activity of protein kinase C (PKC) in cultured human retinal pigment epithelium (RPE) cells in vitro. Methods RPE cells were cultured in standard medium with 10% serum concentrations containing 0.5 to 5.0 ?mol/L hypericin with or without preincubation of phorbol 12 myristate 13 acetate (PMA). The activities of cytosolic PKC (c PKC) and membranous PKC (m PKC) were assayed by PKC kit. Results The original activities of c PKC and m PKC of RPE cells were (35.34?4.10) pmol?min 1 ?mg 1 and (62.52?8.80) pmol?min 1 ?mg 1 . The activity of c PKC in RPE cells with PMA preincubation decreased rapidly in 5 minutes, with a subsequent slow decrease after 20 minutes and a decrease to 18% of the activity of c PKC in RPE cells without PMA preinubation after 60 minutes. While the activity of m PKC in RPE cells with PMA preincubation increased gradually after 5 minutes and reduced after reached the peak at 40 minutes, and then returned to baseline after 60 minutes, eventually decreased below 30% of the control group. When RPE cells were cultured with PMA for 48 hours, the activities of c PKC and m PKC were hardly detectable, while RPE cells were cultured with both PMA and hypericin, hypericin could counteract most of down regulation by PMA. Conclusion Hypericin may inhibit the translocation of PKC in RPE cells,change the activity of PKC, promote the apoptosis of RPE cells likely,and then prevent proliferative vitreoretinopathy.