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Objective:To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells.Methods:SARS-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, GFP1-10 plasmid 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. Results:DNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed that S-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). Conclusion:Overexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.
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AIM: To investigate the occurrence and possible mechanism of blue light-induced ferroptosis in retinal pigment epithelial cells.METHODS: ARPE-19 cells cultured in vitro were irradiated by 405 nm blue light at 50 mW/cm2 irradiance with different duration and were divided into control, 16.3J/cm2, 32.6J/cm2, and 65.2J/cm2 groups; the 65.2J/cm2 group was defined as the high-level blue light irradiation group and cells were further divided into control, high-level blue light irradiation group and high-level blue light irradiation + ferroptosis inhibitor group. CCK-8 assay was used to detect cell viability, commercial kits were used to detect intracellular glutathione(GSH), ferrous iron and malondialdehyde(MDA)concentration, and Western blot was used to detect the relative expression of glutathione peroxidase 4(GPX4)and xCT proteins in cells.RESULTS: The decrease of ARPE-19 cell viability caused by blue light irradiation was dose-dependent, and the reduction of intracellular GSH concentration, the increase of ferrous iron concentration and MDA concentration were all caused by high-level blue light irradiation(all P&#x003C;0.05); the ferroptosis inhibitor partially restored cell viability and recovered intracellular GSH, reduced concentrations of MDA and ferrous iron in the blue light irradiation group(all P&#x003C;0.05). The relative expressions of GPX4 and xCT proteins were significantly decreased in the blue light irradiation group, and such change was alleviated by the ferroptosis inhibitor(P&#x003C;0.05).CONCLUSION: Blue light irradiation may induce ferroptosis in RPE cells by targeting the xCT and GPX4-associated antioxidant pathways.
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@#Age-related macular degeneration(ARMD)is a main cause of irreversible visual impairment in the elderly. The major pathological features are drusen formation, macular pigment disorder, geographic atrophy and abnormal neovascularization. Retinal pigment epithelium(RPE)function is impaired in ARMD. Endoplasmic reticulum(ER)is an organelle in eukaryotes responsible for protein synthesis, modification, integration and quality control. ER also participates in the maintenance of calcium homeostasis and lipid biosynthesis. Stimuli from the external and internal environment may trigger ER stress and therefore activate the intracellular signal transduction pathway-the unfolded protein response(UPR), to restore cell homeostasis. However, prolonged ER stress may lead to apoptosis. The pathogenesis of ARMD has not been fully elucidated, nevertheless, compelling evidence demonstrates that ER stress is involved. In this article, we summarize recent advances in UPR pathways, as well as the role of ER stress in the physiological function of RPE and in the pathogenesis of ARMD.
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Age-related macular degeneration (AMD), the leading cause of central vision loss among people aged 50 years and older, is one of the major eye diseases causing blindness in the world.Clinically, advanced AMD is divided into two types, non-exudative AMD with manifestation of geographic atrophy and exudative AMD with manifestation of choroidal neovascularization.The pathogenesis of AMD is complex, and the para-inflammation is recognized as an important risk factor.Nucleotide-binding oligomerization domain like receptors 3 (NLRP3) inflammasome is a cytoplasmic pattern recognition receptor and is expressed in several kings of cells, including retinal pigment epithelium (RPE) cells, microglial cells, Müller glia cells and retinal vascular endothelial cells.Recent studies have suggested that NLRP3 inflammasome plays an important role in the pathophysiology of both non-exudative and exudative AMD.The role of the NLRP3 inflammasome and its effector cytokines interleukin (IL)-1β and IL-18 in AMD were reviewed in this article to provide guidance on future prevention and therapy of AMD.
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@#Proliferative vitreoretinopathy(PVR)is a serious complication that occurs in the natural history of rhegmatogenous retinal detachment(RRD)or after retinal detachment surgery, often resulting in vision loss. Currently, there has no effective treatment. The pathological characteristics of PVR are the excessive inflammatory response and abnormal proliferation of various cells under the action of cytokines, which eventually form a layer of proliferative membrane around the retinal surface, and further lead to traction retinal detachment(TRD). In-depth studies on the pathogenesis of PVR will help to find promising molecular targets for its treatment. Recent studies have found that vascular endothelial growth factor(VEGF)and the epithelial-mesenchymal transition(EMT)of retinal pigment epithelium(RPE)cells play an important role in the pathogenesis of PVR. This article summarizes the roles of VEGF and RPE cell EMT in the pathogenesis of PVR and the interaction mechanism between them, with the aim to provide new ideas for the treatment and clinical research of PVR.
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AIM:To investigate the effect of epigallocatechin gallate(EGCG)on the apoptosis of human retinal pigment epithelium(ARPE-19)cells and its mechanism. METHODS:The ARPE-19 cells were cultured in vitro and treated with 0,40,80 and 160 μg/mL EGCG, respectively. At the proposed time of treatment the morphological changes were detected by hoechst 33258 staining. The apoptosis rate was detected by flow cytometry. The expression of apoptosis-related factors B lymphocytoma-2 gene(bcl-2), BCL2-Associated X protein(Bax),caspase-3 and p53 were detected by quantitative RT-PCR and Western blotting.RESULTS: Hoechst 33258 staining showed that the ARPE-19 cells with the increase of EGCG drug concentration, the number of apoptotic cells gradually increased and the apoptotic bodies were observed. Flow cytometry showed that the apoptosis rate increased gradually with the increase of EGCG drug concentration. The apoptosis rates at 40, 80 and 160 μg/mL were 4.95%±0.071%, 11.75%±0.075% and 21.25%±0.919% respectively, which was significantly different compared with the control group(2.8%±1.556%)(P<0.01), presented with a drug concentration-dependent. The results of quantitative PCR and Western blotting showed that EGCG could significantly up-regulate the expression of apoptosis-promoting factors Bax, caspase-3 and the mRNA and protein expression of p53, and down-regulate the apoptosis-inhibiting factor bcl-2, all of these showed concentration-dependent effects.CONCLUSION:EGCG can obviously induce the apoptosis of ARPE-19 cells. The mechanism is related with the inhibition of bcl-2 and increase the expression of Bax, caspase-3 and p53.
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@#AIM: To observe the effects of pyridoxamine(PM)on RAGE, ROS and apoptosis in RPE cells treated with advanced glycation end products(AGEs), and to investigate the protective effect of PM on RPE cells in diabetic retinopathy. <p>METHODS:Primary cultured human RPE cells, the third generation of cells were synchronized with serum-free Dulbecco-modified Eagle medium for 24h, and then grouped: 1)Control group: cultured with 100mg/L BSA for 48h; 2)AGEs-treated group: cultured with 200mg/L AGEs for 48h; 3)PM group: PM1 group: cultured with 16mg/L PM+200mg/L AGEs for 48h; PM2 group: cultured with 32mg/L PM+200mg/L AGEs for 48h. The expression of RAGE protein was detected by immunohistochemistry. The formation of ROS was observed by fluorescence microscopy. The apoptosis of cells was detected by TUNEL. <p>RESULTS:The expression of RAGE protein, ROS and apoptosis of RPE cells in PM group were significantly lower than those in AGEs-treated group, and decreased with the increase of PM concentration. <p>CONCLUSION:Pyridoxamine can inhibit the expression of RAGE and the production of ROS, reduce apoptosis, and have a protective effect on RPE cells.
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Objective To observe the effects of resveratrol on human retinal pigment epithelium cells against H2O2-induced cell apoptosis and cell cycle. Methods Human RPE cells were divided into 3 groups ran-domly including normal control group,H2O2injury group and resveratrol protected group. H2O2injury group was treated with 200 μmol/L H2O2,and resveratrol protected group were treated with 50 mg/L resveratrol and then treat-ed with 200 μmol/L H2O2.CCK-8 was used to observe inhibitory rate of cell,and the flow cytometry was applied to detect the apoptosis and cell cycle distribution. Results CCK-8 assay showed that the inhibitory rate of cell treat-ed with resveratrol(50 mg/L)were obviously decreased,which was significantly different with H2O2injury group (P < 0.05),the apoptosis levels of cell treated with resveratrol for 2,8,24 h were significantly decreased(P <0.01).Moreover,the cell cycle distribution in the G0/G1 phases was also significantly decreased(P<0.01),the cell cycle distribution in the S phases was significantly increased(P<0.01).Conclusion Resveratrol can inhibit the cell apoptosis on human retinal pigment epithelium cells induced by H2O2,which is related to cell cycle regula-tion.
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@#AIM: To invesitigate the effect of invigorating blood and dissipating masses traditional Chinese medicine compound drug-containing plasma on the proliferation of rabbit retinal pigment epithelium(RPE)cells treated with platelet derived growth factor(PDGF). <p>METHODS: Primary cells of RPE cells were obtained by enzymatic digestion, and the primitive culture and subculture of RPE cells were proceeded; prepared blood plasma-contained traditional Chinese medicine compound drug-containing plasma; the fourth genertation rabbit RPE cells were selected as the experimental cells by PDGF of low, medium and high doses(5μg/L, 10μg/L, 20μg/L)for 48h. Suitable concentration was detected and selected in cells experiment by using CCK-8 method. Establishing rabbit model of RPE cell proliferation treated with PDGF. The experimental groups were blank control group(DMEM), normal plasma group, PDGF(10 μg/L)group, PDGF(10 μg/L)+ AG1296(10 μmol/L)group, PDGF(10 μg/L)+ 10% drug-containing plasma, PDGF(10 μg/L)+ 20% drug-containing plasma. Respectively, transwell method was used to determine the migration of rabbit RPE cells after 24h intervention in each group; CCK-8 was used to determine the cell viability OD value of rabbit RPE cells after 48h of intervention in each group.<p>RESULTS: The plasma containing 10% and 20% concentration of invigorating blood and dissipating masses traditional Chinese medicine compound drug can effectively inhibit cell viability and cell migration of RPE cells treated with PDGF. <p>CONCLUSION: We found the certain concentration of invigorating blood and dissipating masses traditional Chinese medicine compound drug can effectively inhibit cell viability and cell migration of RPE cells treated with PDGF, which may be an effective treatment for proliferative vitreoretinopathy and provide a new way to study the mechanism of proliferative vitreoretinopathy.
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·AIM: To investigate the effect of Ghrelin on oxidative stress induced by high glucose in human retinal pigment epithelium (RPE) cells. ·METHODS: RPE cells were cultured and divided into the negative control group, high sugar group, Ghrelin low dose group ( 10-9mol/L ) and high dose group (10-6mol/L). Cells survival rate were detected by CCK-8 colorimetry, cells oxidative damage were observed by oxygen sensitive fluorescence probe H2DCFDA staning, changes of intracellular reactive oxygen species ( ROS ) were detected by H2DCFDA staining, super oxide dismutase ( SOD) activity and malondialdehyde ( MDA) content were detected by spectrophotometer colorimetry. ·RESULTS: CCK-8 results showed that RPE cells survival rate increased to 54.79%±3.43% and 79.16%±3.29% after treated with 10-9mol/L, 10-6mol/L Ghrelin, the difference was statistically significant compared with high glucose group (41.65%±3.42%)(P<0.05). H2DCFDA fluorescent probe dying showed that Ghrelin reduced ROS generation in RPE cells and decreased oxidative damage cells. Spectrophotometer colorimetric method showed that according to the high sugar group, SOD activity increased and MDA content decreased in Ghrelin group. ·CONCLUSION: Ghrelin could inhibit high glucose - induced oxidative damage in human RPE cells, which has protective effect on the process of the occurrence and development of diabetic retinopathy.
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Background Retinal pigment epithelium (RPE) cell transplantation is the primary means of human trial for the treatment of retinal degeneration.Induced pluripotent stem cells (iPSCs) will become an important source for cell transplantation.In addition,iPS-RPE cells may provide a personalized treatment platform for the patient's own cells treatment.Objective This study was to evaluate the feasibility of human fibroblasts differentiate toward iPSCs from retinitis pigmentosa (RP) patients and toward iPSC-RPE cells from non-RP individual by retroviral transfection of Oct4,Sox2,c-Myc and KLF4 genes.Methods Human thigh skin tissues were obtained from a RP patient with hotspot mutation of SNRNP200 p.S1087L and individual without SNRNP200 p.S1087L mutation,respectively,with the size 2 c m×3 cm.Human dermal fibroblasts were isolated and cultured by trypsin digestion and explant method.The fibroblasts were transfected by a series of retrovirus and cultured by human embyonic cellconditioned medium to generate and induce iPSCs,and then the iPSCs were identified by morphology,alkaline phosphatase (AP) staining and immunofluorescence assay of specific markers of pluripotent stem cells.iPSCs suspension were injected into SCID mouse to observe the tumorigenesis.The iPSCs from non-RP subject were induced to differentiate toward iPS-RPE cells by embryonic body (EB) inducing method,and iPS-RPE cells were identified by detecting the expression of RPE65,zonula occludens protein 1 (ZO-1) and lecithin retinol acyltransferase (LRAT).Results Cultured human dermal fibroblasts showed fusiform or polygon shape and intercellular close arrangement,and Vimentin was positively expressed in the cells.Small cell colonies were harvested 5-7 days after infected by retroviruses,and the morphology changed from spindle into round mass.The hESC-like iPSCs clonies appeared 20 days after cultivation,and the positive expressions of hESC-specific surface antigens including SSEA3,SSEA4,TRA-1-60,TRA-1-81 and Nanog were found in the cells 25-30 days after cultivation,and the positive staining of AP was obtained 12 weeks after cultivation.A teratoma was formed at the injection site of iPSCs suspension in SCID mouse.Immunofluorescence technique showed that RPE cell-specific proteins including RPE65,ZO-1 and LRAT proteins were positively expressed in iPS-RPE cells at 30 days after differentiation.Conclusions Mutation SNRNP200 p.S1087L of RP patient-specific iPSCs can be induced from human dermal fibroblast by retrovirus infection method.The function and morphology of the iPSCs were similar to hESCs.Human iPSCs cell line generated from the dermal fibroblasts of non-RP individuals can differentiate into iPS-RPE cells.
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Objective To investigate the transfection efficiency of combination of ultrasound (US) microbubbles (MBs) mediated liposome small interference RNA (siRNA) to human and rat retinal pigment epithelium (RPE) cells. Methods Human and rat RPE cells and siRNA were incubated in 24-well plates (2×10~5/well and 1×10~5/well, respectively). The cells were devided into 5 groups: siRNA+US, siRNA+MBs+US, siRNA+L (liposome), siRNA+L+US, siRNA+L+US+MBs. After 12 h, transfection efficiency was observed with fluorescence microscopy and flow cytometry. Results US or ultrasound targeted microbubbles destruction without liposome-mediated could not promote siRNA transfection efficiency to human and rat RPE cells. The transfection efficiency of human and rat RPE cells significantly decreased in the siRNA+L+US+MBs group, but increased in siRNA+L+US group. Conclusion Ultrasonic irradiation can promote lipid-mediated siRNA transfected human RPE cells.
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Background Culture of retinal pigment epithelium(RPE) cells is very important for establishment of proliferative vitreoretinopathy (PVR) model,prevention and treatment of PVR as well as RPE cell transplantation.Isolation of animal RPE cells by trypsinization is a critical step.ObjectiveThe present study is to establish the methods of isolation and culture of retinal pigment epithelium (RPE) cells in rabbit and comparied with that of pig RPE culture.MethodsRPE cells were isolated by trypsinization in pigmented rabbit and pig and cultured in DMEM containing 20% fetal bovine serum.Cultured RPE cells were identified by immunochemistry.The fourth generations of cells were used in this experiment.Morphology and characteristics of cultured RPE cells from rabbit and pig were examined and compared under the light microscope.ResultsIsolated RPE cells from pig were obtained by once trypsin digestion,but two times of trypsinization were needed in rabbit RPE cells isolation.The differentiation in response to trypsinization was related to anatomic difference between the two types of cells .The adherence time of pig RPE cells was 24 hours ,however,the rabbit RPE needed 48-72 hours after culture.Proliferation and vitality of cultured cells were gradually attenuated and melanin decreased after several times subculture.The morphology of culture RPE cells was obviously different between rabbit and pig because species difference.Immunohistochemistry demonstrated the positive response of RPE cells for keratin.ConclusionRPE cells can be acquired from both rabbit and pig by trypsinization and culture.The culture process of RPE cells of pig is simpler than that of rabbit.Cells within the fourth generations are suitable for experimental application.
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To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on the proliferation and PCNA (proliferating cell nuclear antigen) expression of cultured human retinal pigment epithelium cells, human retinal pigment epithelium cells (RPE) were cultured from normal adults who died accidentally. The effects of PDTC on the proliferation of RPE cells were examined by using methyl thiazlyl tetrazolium (MTT) assay. The effects of PDTC on the PCNA expression of RPE cells were immunohistochemically examined by employing biological image analysis system (BIAS). After treatment with PDTC of various of concentration ranging from 0.062 to 1 g/L for 24 h, or concentrations ranging from 0. 031 to 1 g/L, the proliferation of RPE cells decreased in a dose-dependent manner. After treatment with PDTC of concentration varying from 0. 062 to 1 g/L for 24 h, the PCNA expression was also suppressed in a dose-dependent manner. It is concluded that PDTC can inhibit the proliferation of RPE cells in vitro in a dose-and time-dependent manner, at least in part,by down-regulating the expression of PCNA. PDTC may be used to prevent and treat the proliferative vitreoretinopathy (PVR).