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Background Stem cell transplantation represents a promising treatment option for patients suffering from degenerative disorders.Accumulating evidences indicate that mesenchymal stem cells (MSCs) are able to differentiate into retinal pigment epithelial (RPE)-like cells.However,MSCs are difficult to obtain.Human adipose mesenchymal stem cells (ADSCs) are proved to have similar properties to MSCs,but relevant study is less.Objective This study was to assess the feasibility of human ADSCs differentiating into RPE-like cells and the safety of its application in vivo.Methods The third generation of human ADSCs were incubated into 6-well plate,and 100 ng/ml epithelial growth factor,50 μ mol/L taurine and 5×10-7 mol/L retinoic acid were added into the medium 12 hours after cultured to induce the cells,and conventional cultured cells were used as the control group.Induced cells were traced with PKH26,and Pan-cytoke ratin (Pan-CK) monoclonal antibody was used to identify the cells under the fluorescence microscope.Induced RPE-like cell suspension of 1 μl was intravetreally injected in the right eyes of 6 BALB/c mice,and equal volume of PBS was used in the same way in another 6 mice.The animals were sacrificed 1 month after injection,and the retinal morphology was examined by histopathology under the optical microscope.The ultrastructure of retinal ganglion cells (RGCs) was examined by the transmission electron microscope.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.Results Cultured human ADSCs grew well with the slender polygone shape.Cell membranes showed the red fluorescence for PKH26 after induced.In addition,Pan-CK was expressed in the cell membranes with the red fluorescence in the induced cells,but the response was absent in the control cells.One month after intravitreal injection,induced cells located on the retinal surface,and the retinal morphology was clear under the optical microscope.No abnormality in RGCs was seen under the transmission electron microscope.Conclusions Human ADSCs can differentiate into RPE-like cells after induction.PKH26 can mark induced cells well.There is no adverse effect of induced cells on retina after intravitreal injection in a short-term duration in mice.
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Background Researches showed that stem cells can rescue damaged cells through mitochondrial transfer.This mode has been used to regenerative cell-based therapy.Retinal pigment degeneration is an eye disease of retinal pigment epithelial (RPE) cell apoptosis as pathogenetic mechanism.Whether stem cells can repair the target cells by above mechanism has not been clarified.Objective This study was to investigate the influence of mitochondrial transfer on the function of RPE cells.Methods The RPE cells of Long-Evans rats were isolated and cultured and the third generation of cells were used in sequential experiment.The cells were identified by detecting the expressions of RPE65 and Bestrophin proteins with immunofluorescence stain.Mouse neural stem cells (NSCs) (C17.2 strain) with green fluorescence protein (GFP) and without GFP were cultured.Mitotracker-green and Mitotracker-red staining were separately used to labeled the mitochondria of RPE cells and NSCs.RPE cells were cocultured with NSCs,and wheat germ agglutinin (WGA) was used to mark the tunneling nanotubes (TNT) between the two kinds of cells,and then the mitochondrial migration in TNT was exhibited by the laser scanning confocal microscope.The proportion of RPE cells in different cycles was assayed after marked with propidium iodide (PI) by flow cytometry.The contents of ATP,ADP and AMP in RPE cells were detected by high performance liquid chromatography (HPLC).Results The third-generation of RPE cells grew well with the RPE65-and Bestrophinpositive rate ＞85％.The Mitotracker-red-labeled rates of NSCs and RPE cells were no less than 95％.TNT structure was seen to appear the blue fluorescence between RPE cells and NSCs 24 hours after co-cultured and the red dye mitochondria from NSCs migrated toward red dye mitochondria from RPEs with the lapse of time.The RPE cell proportion reduced in G1 phase and increased by 5％ and 2％ in the S phase and G2/M phase respectively after mitochondrial transfer than before (P=0.016,0.114,0.189).The contents of ATP,ADP and AMP in the RPE cells were (8.77 ±3.68),(2.76±0.92) and (1.07 ±0.65) μg/mg after cell co-culture,and those before co-culture were (11.29±2.29),(3.12±0.95) and (1.59± 1.22) μg/mg,without significant differences between them (P =0.370,0.668,0.553).Conclusions NSCs can transfer normal mitochondria to co-cultured RPEs via TNT structure.Mitochondrial exchange might be one of therapeutic mechanisms of NSCs recuing damaged RPE cells.
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Background Researches showed that platelet-derived growth factor (PDGF) modulate the expression of matrix metalloproteinase/tissue inhibitor of metalloproteinase (MMP/TIMP) in cells,but the association of expression of MMP/TIMP in retinal pigment epithelial (RPE) cells and the dose and active time of PDGF is unclear.Objective This study was to observe the effects of PDGF on the expressions of MMP-2,MMP-9 and TIMP-1 in cultured RPE cells in vitro.Methods RPE cell line,ARPE-19,was calculated in vitro,and the cells were divided into 5 groups when they reached 70％-80％ confluence.Different concentrations (0,0.1,1,10,50 mg/L) of PDGF was added into the medium respectively for 36 hours,and the expressing levels of mRNA and protein of MMP-2,MMP-9 and TIMP-1 were detected by reverse transcription PCR (RT-PCR) and Western blot assay.In addition,RPE cells in PDGF group were treated with 10 mg/L PDGF for 24,36,48 hours respectively to detect the expressions of mRNA and protein of MMP-2,MMP-9 and TIMP-1 in the cells and to compare with the control group without PDGF.Results PDGF stimulated proliferation of RPE cells in a dose-and time-dependent manner.As the increase of the PDGF concentrations,the expression values of MMP-2 mRNA and MMP-9 mRNA in RPE cells were gradually elevated,with a statistically significant difference among various groups (MMP-2 mRNA:F=79.304,P=0.000;MMP-9 mRNA:F =8.465,P=0.003),and the expressions of MMP-2 mRNA and MMP-9 mRNA were significantly higher in the 1,10,50 mg/L PDGF groups compared with 0 mg/L PDGF normal control group (all at P＜0.05).Also,the expression values of MMP-2 and MMP-9 proteins in RPE cells were gradually elevated with the increase of PDGF concentrations,showing statistically differences among the groups (MMP-2:F=26.550,P=0.000;MMP-9:F=80.993,P=0.000).Compared with the 0 mg/L PDGF group,MMP-2 and MMP-9 expression levels in the 1,10,50 mg/L PDGF groups were significantly up-regulated (all at P＜ 0.05).However,the expression levels of TIMP-1 mRNA and protein group in the cells were not significantly different among various groups (mRNA:F =0.143,P =0.962 ; protein:F =1.955,P =0.178).The expression levels of M MP-2 mRNA,M MP-9 mRNA in the cells were increased in the PDGF group compared with the control group at different time points (MMP-2 mRNA:Ftime =83.250,P=0.002 ; MMP-9 mRNA:Ftime =6.785,P =0.019).Also,the expression values of MMP-2 and MMP-9proteins in RPE cells were increased in the PDGF group compared with the control group at different time points (MMP-2:Ftime =1 l.185,P =0.041 ; MMP-9:Ftime =968.413,P =0.000).The expression levels of MMP-2 and MMP-9 mRNAs and proteins were significant between the two groups at different time points (all at Pgroup =0.000;all at Ptime＜0.05).While the expression changes of TIMP-1 werc not significant between the two groups and among various time points (all at P＞0.05) Conclusions PDGF up-regulates MMP-2 and MMP-9 expressions in RPE cells in a dose-and time-dependent manner.But,PDGF dose not alter the expression of TIMP-1.These results indicate that PDGF disrupt the balance of MMP/TIMP,which may damage the extracellular matrix and therefore facilitate the migration of RPE cells in the pathogenesis of proliferative vitreoretinopathy.
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ObjectiveTo observe the effect of visible light (white light, red light, blue light) on the expression of reactive oxygen species (ROS), 8-OHdG and hOGG1 in cultured human retinal pigment epithelial (RPE) cells. MethodsCultured human RPE-19 cells (4th-6th generations) were divided into white light,red light, blue light and control group.The illumination was 600 Lux.The cells of experimental groups were exposed to white light or red light for 6, 12, 24 and 48 hours, and exposed to blue light for 1, 3, 6 and 12 hours, while cells of the control group were cultured in foil packaged dishes to avoid light. The levels of ROS expression were detected by 2′, 7′-dichlorofluorescin-diacetate (DCFH-DA), the levels of 8-OHdG protein expression were observed by immunocytochemistry (ICC), and the levels of hOGG1 were measured by western blot. ResultsCompared to the control group, the ROS expression in RPE cells were increased in white and red light group after 12, 24 and 48 hours and in blue light group after 1, 3, 6 and 12 hours (Fwhite light=11. 611, Fred light =6.706, Fblue light =23. 259; P＜0.05 ). Additionally, the ROS expression had a tendency to increase gradually along with exposure time. Compared to the control group, the 8-OHdG expression in RPE cells were increased significantly in both white and red light group after 12, 24 and 48 hours and in blue light group after 1, 3, 6 and 12 hours (Fwhite light =16. 032,Fred light =6. 378, Fblue light =19. 484; P＜0.05). Additionally, the 8-OHdG expression in white and red light group were increased gradually with exposure time but decreased when exposure time was up to 48 hours, while that in blue light group was increased firstly though it started to decrease when exposure time was up to 6hours. Compared to the control group, the hOGG1 expression in RPE cells were increased in white and red light group after 12, 24 and 48 hours and in blue light group after 6 and 12 hours (Fwhite light =15. 121,Fred light=21. 041,Fblue light12. 479; P＜0.05). ConclusionsExposure to white, red or blue light could induce ROS production and DNA oxidative damage in RPE cells in a time-dependent way.Exposure to visible light could switch on self protection of RPE cells against DNA oxidative damage by up-regulating of the hOGG1 expression.
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Objective To investigate the relationship between exposure intensity and illumination time of blue light and replicative senescence of rat retinal pigment epithelial (RPE) ceils.Methods Thirty-six 12-14 weeks Wistar rats were kept in the cage with a blue-light bulb [(450±10) nm],and were randomly divided into four groups (no light,nature light,500 lx light and 1000 lx light illumination),each has nine rats.The rats in each group were further divided into three subgroups according to illumination time (one month,two months or three months).Eyeballs were collected after intraperitoneal injection of 10% chloral hydrate.The right eye of each rat was embedded in paraffin and sectioned for hematoxylin-eosin (HE)staining,while frozen sections of the left eye were stained for the senescence-associated β-galactosidase (SA-β-Gal).The data were analyzed by SPSS11.5 statistical software.Results The amounts of SA-β-Gal positive RPE cells were significantly different between all groups under the same illumination time 17 (P=0.000),and between all subgroups of different illumination time with same exposure intensity (P＜0.01)except for the control group (no light).Conclusion Blue-light can induce replicative senescence in rat RPE cells in an intensity and time-dependent manner.
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Objective To investigate the feasibility of differentiation of invitro induced rat bone marrow-derived mesenchymal stem cells(rMSCs) into retinal pigment epithelial (RPE) cells.Methods The rMSCs from Brwon-Norway (BN) rats were isolated and cultured by adherent screening method.RPE cells lysate made by repeated freeze-thawing was put into the rMSCs culture system to identify whether the induced cells could express characteristic label cytokeratin(CK)and S-100 simultaneously or not.Results The growth rate of rMSCs induced by RPE cells lysate was slower and protuberant burr surrounded the fusiform cells.The results of immunoblotting and double immunofluorescence showed that partial induced cells expressed CK and S-100 simultaneously.The result of flow cytometry indicated that 14.1% induced cells expressed CK and S-100 simultaneously.Conclusion Induced by RPE cells lysate,rMSCs can differentiate into RPE cells.
