Transcriptomics analysis of hypoxia-induced retinal pigment epithelium cell injury / 中华实验眼科杂志

Objective:To analyze differentially expressed genes (DEGs) and the changes of signal pathways in human retinal pigment epithelium cells (ARPE-19) under hypoxic and normoxic conditions and to explore the biological mechanism of hypoxia-induced ARPE-19 cell damage via transcriptome sequencing (RNA-seq) and bioinformatics technology.Methods:The ARPE-19 cells were divided into the hypoxia treatment group and the normoxia control group treated with 1% and 21% O 2 by volume for 8, 24, 48, 72 hours, respectively.The relative expression levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) mRNA were detected with real-time fluorescent quantitative PCR at different time points.RNA-seq and bioinformatics analysis were performed at 8 hours and 24 hours after hypoxia and normoxia treatment.DEGs were screened out under the conditions of |log 2FC|≥1 and P≤0.05.Then the cluster heat map analysis, Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis and protein-protein interaction network analysis were also carried out.Real-time fluorescent quantitative PCR was employed at 24 hours after hypoxia to detect the relative mRNA expression of genes that might be related to hypoxia in DEGs.Cell viability kit was used to verify and compare the damage effect of hypoxia on ARPE-19 cells at different time points between the two groups. Results:The relative mRNA expression levels of VEGF at 8, 24, 48 and 72 hours after hypoxia treatment and the relative HIF-1α mRNA expression levels at 8, 24 and 48 hours after hypoxia treatment were significantly higher than those of the normoxia control group (all at P<0.05). There were large differences in the mRNA expression levels at 8-hour and 24-hour treatment between the two groups.A total of 62 significant DEGs were screened between the hypoxia treatment group and the normoxia control group after 8-hour hypoxia treatment, among which 45 genes were significantly up-regulated and 17 genes were significantly down-regulated.A total of 255 significant DEGs were screened out between the hypoxia treatment group and the normoxia control group after 24-hour hypoxia treatment, among which 228 genes were significantly up-regulated and 27 genes were significantly down-regulated.The GO functional analysis of DEGs was mainly enriched in processes such as protein degradation, nucleotide biosynthesis, and material transport.KEGG pathway analysis was mainly enriched in PI3K-Akt, cGMP-PKG, and other signaling pathways closely related to metabolism, cell cycle, cell growth, and apoptosis.The core genes HPCA, MT3 and NOS3 were found by protein-protein interaction network analysis.Real-time fluorescent quantitative PCR test results showed that after 24-hour hypoxia treatment, the mRNA expression levels of hypoxia related genes DEPP1, NPPB, PDZK1, HILPDA, TCEA3, NDRG1 and RORC in ARPE-19 cells were significantly increased and the mRNA expression levels of TFRC and NQO1 were significantly decreased (all at P<0.05). The cell morphology was normal and the growth state was good without dead cells after 8-hour and 24-hour hypoxia treatment in ARPE-19 cells.There were dead cells after 48-hour hypoxia treatment, and the number of dead cells was increased at 72 hours after hypoxia treatment. Conclusions:The PI3K-Akt and cGMP-PKG signaling pathways related to metabolism may be involved in hypoxia-induced injury of ARPE-19 cells.Core genes of HPCA, MT3 and NOS3 can be used as functional target genes and play key roles in hypoxia response of cells.

Effects of lutein on transforming growth factor-β2 induced epithelial-mesenchymal transition in ARPE-19 cells / 上海交通大学学报（医学版）

Objective • To establish the transforming growth factor-β2 (TGF-β2) induced epithelial-mesenchymal transition (EMT) model of retinal pigment epithelium cells, and investigate the effect and mechanism of lutein on EMT. Methods • ARPE-19 cells were cultured and divided into 4 groups including control group, TGF-β2 group, TGF-β2+lutein group and lutein group. The mRNA levels of α-smooth muscle actin (α-SMA), fibronectin (FN) and E-cadherin were analyzed by real-time PCR. The protein expression of α-SMA, FN and occludin were assayed by Western blotting. Immunofluorescence was used to detect the change of α-SMA. Meanwhile, Western blotting was performed to detect the expression levels of pSmad3 in the TGF/Smad signaling pathway. Results • TGF-β2 induced EMT was inhibited by lutein. Lutein decreased the mRNA and protein levels of the mesenchymal markers α-SMA and FN, and increased the expression of the epithelial markers E-cadherin and occludin (all P<0.05). Immunofluorescence showed that lutein can inhibit the conversion of epithelial cells into myofibroblasts. Lutein significantly downregulated the high expression of pSmad3 in TGF-β2 treated ARPE-19 cells (P=0.001). Conclusion • Lutein inhibits TGF-β2 induced EMT by downregulating the expression of pSmad3 in TGF-β/Smad signaling pathway, indicating it may attenuate subretinal fibrosis.

Role of mitochondrial DNA in human retinal pigment epithelium cells apoptosis induced by blue light / 中华实验眼科杂志

Objective To research the role of mitochondrial DNA mediate the cultured human retinal pigment epithelium (hRPE) cell apoptosis induced by blue light and the relationship with time.Methods Established the blue light damage model of cultured hRPE cells in vitro with light emitting diode (LED) blue light density of (4.0-±0.5)mW/cm2 adjusted by FL-1D blue light illumination meter,and the illumination time was set as 0,0.5,1,2,4,6,12 and 24 hours,then the cells were grouped according to the illumination time.Immunofluorescence were used to identify the cells;the expressions of caspase-3,cleaved caspase-3,caspase-9,cleaved caspase-9,bax and bcl-2 were detected with Western blot.Quantitative PCR was used to detect the copy number of mitochondrial DNA and PCR was used to detect mitochondrial DNA 4977bp common deletion.Results Immunofluorescence results showed that the RPE65 protein was expressed in the cytoplasm.The expressions of bax were upregulated after illumination for 1 hour,cleaved caspase-3 were upregulated after illumination for 2 hours,caspase-3,caspase-9,cleaved caspase-9 were upregulated after illumination for 4 hours,while the expression of bcl-2 was downregulated after illuminated for 2 hours,with significant differences compared to the normal control group (all at P＜0.05).The copy number of mitochondrial DNA in 0.5,1,2,4,6,12 and 24 hours groups was downregulated,with significant differences compared to the normal control group (all at P＜0.05).The expressions of 4977bp common deletion in 0.5,1,2,4,6,12 and 24 hours groups were increased,with significant differences compared with the normal control group (all at P＜0.05).Conclusions Blue light can cause cell apoptosis,especially mitochondrial apoptosis,in hRPE probably motivated by mitochondrial DNA damage.

Expression of VEGF165 and VEGF165b in human retinal pigment epithelial cells in hypoxia and high glucose environment / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM:Through the expression of VEGF165and VEGF165bin human retinal pigment epithelial cells in vitro in artificial simulated hypoxia and high glucose environment, to discuss their roles in the development of diabetic retinopathy and the relationship between each other. ·METHODS: After normal inoculation and cultivation of human retinal pigment epithelial cells (RPE) in vitro, the cells was divided into the normal group (5. 56mmol/L glucose,without CoCl2),the hypoxia group(5.56mmol/L glucose + 150μ mol/L CoCl2), the high glucose group (25mmol/L glucose, without CoCl2), the combination group (25mmol/L glucose + 150μ mol/L CoCl2),a total of four groups. The RNA of each group was extracted respectively in 12h,24h,36h,and 48h. We used the MTT colorimetry to detect cell vitality and growth trend; RT-PCR method to detect VEGF165and VEGF165brelative expression of mRNA of RPE cells in four different time points. ·RESULTS:Hypoxia and high sugar environment limited proliferation of RPE cell division and cell vitality. After comparing cells of the same group in different time points, in the normal group there was no statistically significant different expression over time (P>0.05); the expression in the hypoxia group, the high glucose group and the combination group increased over time, the difference was statistically significant(P<0. 05). At the same time,differences of the expression between groups was not statistical significant in 12h (P > 0. 05); the difference was statistically significant in 24h,36h,48h(P<0.05). ·CONCLUSION: Cultured RPE cells can express VEGF165b normal. Lack of oxygen and high glucose can induce the increase of VEGF165 mRNA, at the same time reduces the VEGF165bmRNA expression.

Influence of oxidative stress-induced exosomes on Akt and vascular endothelial growth factor-A of retinal ;pigment epithelium cells / 中华眼底病杂志

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 2.5μmol/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.

Inhibitory effects of autophagy inhibitor 3-MA on high glucose-induced proliferation of human retinal pigment epithelium cells / 中华实验眼科杂志

Background Diabetic retinopathy (DR) is one of the common complications of diabetes.Retinal pigment epithelium (RPE) cells,as important constituent cells of the retina,play important roles in the development and progression of DR.Objective This study was to investigate the suppressive effects of autophagy inhibitor 3-MA on the proliferation of human retinal pigment epithelium cells (hRPECs) following high glucose culture.Methods HRPECs were divided into control group,high-glucose group and 3-MA+high glucose group.The cells were cultured by the DMEM/F12 with 5 mmol/L glucose in the control group,and by the DMEM/F12 with 5 mmol/L glucose in the high glucose group and by the DMEM/F12 with 10 mmol/L 3-MA (for 1 hour firstly) and 30 mmol/L glucose in the 3-MA+high glucose group.The cells were inoculated into 24-well plate with the content of 1 ×105/well,and then the cells were consecutively cultured for 24 hours with DMEM/F12 containing 0.5％ fetal bovine serum after achieved attached 80％ confluence.The morphology and uhrastructure of the cells were examined by optics microscope and transmission electronic microscope.The proliferative rate of the cells was assayed by CCK-8 kit.The expression of autophagy-related gene microtubule associated protein light chain 3B (LC3B) in the cells was detected by Western blot and LC3B-Ⅱ/LC3B-Ⅰ value was quantitatively evaluated among the groups.Results The cells grew well with uniform size and regulatory arrangement in the control group.The cells in the high glucose group enlarged and the number of cells evidently increased.In the 3-MA+high glucose group,the cells decreased with a disorder arrangement.Under the transmission electron microscope,the cells were normal with the round-or oval-like nucleus and normal organelles in the control group,and autolysosome could be seen in the cells in the high glucose group.In the 3-MA+high glucose group,some autophagic bodies were found.The proliferative rate of the cells was (100.0±2.0) ％,(116.9-±5.2)％ and (103.7 ±4.7)％ in the control group,high glucose group and 3-MA+high glucose group respectively,showing a significant difference among the groups (F =13.526,P =0.006).The proliferative rate was considerably raised in the high glucose group compared with the control group and 3-MA+high glucose group (both at P＜0.05),but there was no significant difference in the proliferative rate of the cells between the control group and 3-MA+high glucose group (P＞0.05).Compared with the control group,the expressing intensity of LC3B-Ⅰ protein was weakened and that of LC3-Ⅱ protein was enhanced,and the expression intensity of LC3B-Ⅰ and LC3B-Ⅱ proteins in the 3-MA+high glucose group was similar to that in the control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.131 ±0.065,2.504±0.097 and 0.274±0.007 in the control group,high glucose group and 3-MA+high glucose group,respectively,with significant differences among the groups (F =1 694.676,P =0.000),and the LC3-Ⅱ/LC3-Ⅰ ratio was increased in the high glucose group in comparison with the control group and the 3-MA+high glucose group (all at P＜0.05).No significant difference was found in the LC3B-Ⅱ/LC3B-Ⅰ ratio between the control group and 3-MA + high glucose group (P ＞ 0.05).Conclusions High glucose culture of hRPECs can activate autophagy process and promote cell proliferation.3-MA,an autophagy inhibitor,suppresses the high glucoseinduced growth of HRPECs by inhibiting autophagy process.

Epithelial-mesenchymal transition of human retinal pigment epithelial cells under the high glucose condition in vitro / 中华实验眼科杂志

Background Several types of cells participate in the formation of proliferative membrane in proliferative retinopathy (PVR),and the proliferation,migration and epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) cells play an important role.Many studies have confirmed high blood glucose is the basic pathogenesis of diabetic retinopathy (DR).However,whether EMT could be induced in RPE cells under the high glucose condition has not been reported.Objective This study was to investigate the effects of high glucose on the migration and EMT of RPE cells in high glucose culture model in vitro.Methods Human RPE cell line D407 were cultured and passaged in DMEM/F12 medium with 10％ fetal bovine serum,and 6-8 generations of cells were used in experiment.The cells were divided into 3 groups based on different glucose concentrations in medium.The glucose at the final concentration 5.5 mmol/L or 60.0 mmol/L was respectively used in the normal control group or high glucose group,and the DMEM with 5.5 mmol/L glucose and mannitol was used in the hypertonic control group.The migration rate of the cells were detected 0,24,48 and 72 hours after scratching by wound-scratch test.Real-time PCR was used to detect the relative expressions of zonula occludens-1 (ZO-1) and α-smooth muscle actin (α-SMA) in the cells.Results Cultured cells showed a polygon shape with the clear nucleolus and dense arrangement in the normal control group and the hypertonic control group,but the cells were larger and elongated with the lapse of culture time with the indistinct structure and loose arrangement.At 48 hours after scratching,migrating cells were seen in the scratching area,and the scratching area disappeared at 72 hours after scratching in the high glucose group,but the scratching area still was existed in the normal control group or hypertonic control group.The migrating rate of the cells was higher in the high glucose group than that in the normal control group or hypertonic control group,showing total differences among 3 groups and various time points (Fgroup =328.600,P =0.000 ; Ftime =773.270,P=0.000).Compared with the normal control group,the expression level of ZO-1 mRNA was significantly lower,and α-SMA mRNA level was higher 48 hours and 72 hours in the high glucose group than those in the normal control group (all at P＜0.05).Conclusions High glucose induce the migration and EMT of RPE cells in vitro,which may be associated with the pathogenesis of proliferative diabetic retinopathy.

Oxidative damage of human retinal pigment epithelium cells induced by blue light irradiation and mitochondria-participated mechanism / 中华实验眼科杂志

Background Researches showed that mitochondria and oxidative stress play a crucial role in retinal photochemical injury,but the relationship between the damage of human retinal pigment epithelium (RPE) cell-induced by blue light and light-irradiated time is less studied.Objective The aim of this study was to research the possible mechanism of RPE oxidative damage induced by blue light in vitro.Methods Human RPE cells were isolated from healthy donors and cultured.The cells were divided into the normal control group and the light exposure group.The cells of light exposure group were irradiated using the blue light of (4.0±0.5) mW/cm2 for 0.5,1,2,3,4,5,6,12 and 24 hours,respectively,and the cells of the normal control group were cultured in dark environment.Cellular viability was detected by MTT method,and the ultrastructure change of subcellular organelles in RPE cells was examined under the transmission electron microscope (TEM).The content of reactive oxygen species (ROS) was assayed by flow cytometry for the assessment of oxidative stress reaction.The relative expressions of nicotinamide adenine dinucleotide phosphate (NADPH) mRNA and cyclooxygenase 1 (COX1) mRNA in the cells were detected by real-time fluorescence quantitative PCR to evaluate the mitochondria function.Results The percentages of cellular viability were (100.00±20.00) ％,(95.73±0.89) ％,(94.67±2.56) ％,(84.23±0.16) ％,(78.57±3.09)％,(75.43±2.18)％,(66.13±1.42)％,(53.43±1.91)％ and (47.97±1.36)％ in the normal control group and light exposure for 1-hour,2-hour,3-hour,4-hour,5-hour,6-hour,12-hour and 24-hour groups,respectively,showing a significant difference among the groups (F =172.270,P =0.000),and the percentages of light exposure for the more than 3 hours groups were significantly lower than those of the normal control group (all at P＜ 0.05).The vacuoles-like degeneration,mitochondrial swelling,decreased microvilli were seen under the TEM.The contents of ROS in RPE cells were (14.75±2.49)％,(19.04± 1.02) ％,(22.81 ±3.20)％,(28.75±2.15)％,(33.06±0.96) ％,(40.64±2.11) ％,(48.25±2.50) ％ and (60.44±2.68) ％ in the normal control group and light exposure for 0.5-hour,1-hour,2-hour,3-hour,4-hour,5-hour,6-hour groups,and with significant increases in ROS contents in various light exposure groups compared with the normal control group (all at P＜0.05).The relative expression levels of NAPDH mRNA in the cells were gradually elevated 3 hours after light exposure with the increase of time in comparison with the normal control group (all at P＜0.05),and the relative expression levels of COX1 mRNA in the cells were higher in the light exposure for 2-hour,3-hour,4-hour and 5-hour group compared with the normal control group (all at P＜0.05),and after that the COX1 mRNA levels were gradually declined and were close to the normal level.Conclusions Blue light irradiation for more than 3 hours causes oxidative stress damage of mitochondria in RPE in vitro,and the damage was more obvious after irradiation for 5-6 hours.

Effect of hypoxia and hyperglycemia on retinal pigment epithelial cells co-cultured with human mesenchymal stem cells / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To evaluate cell proliferation, apoptosis and migration of human retinal pigment epithelial cells ( RPE) when co - cultured with human marrow mesenchymal stem cells ( hMSCs ) in condition of hypoxia and hyperglycemia so as to explore possible mechanisms of diabetes aggravating choroidal neovascularization ( CNV) preliminarily. METHODS:Both hMSCs and RPE cells were co-cultured in a transwell system. The experiment was divided into four groups: 21% O2 with 5. 56mmol/L glucose ( control group, A ), 21% O2 with 30mmol/L glucose ( hyperglycemia and normoxia group, B ) , 5% O2 with 5.56mmol/L glucose ( normoglycemia and hypoxia group, C ) and 5% O2 with 30mmol/L glucose ( hyperglycemia and hypoxia group, D) . Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of RPE cells in each group at 12, 24 and 48h respectively. Flow cytometry was performed to observe apoptosis of RPE cells at 24h. Additionally, we assessed migration capabilities of RPE via transwell assay under the condition of hyperglycemia and hypoxia by co-culturing of hMSCs.RESULTS:In this co-culturing system, at 12, 24 and 48h, group B (1. 61±0. 41, 1. 80±0. 34;1. 91±0. 35), C (1.34±0. 46, 1. 94±0. 40, 2. 14±0. 41) and D (1. 98±0. 47, 2.26±0.42, 2. 55±0. 40) showed significantly higher proliferation rate than group A (0. 92±0. 45, 1. 27±0. 32, 1.59±0. 41, P0. 05). CONCLUSION:By coexistence with hMSCs, the synergy of hyperglycemia and hypoxia can improve migration and proliferation of RPE cells, and have no effect on apoptosis of RPE cells within short period.

Research progress in autophagy on dry age-related macular degeneration / 中华实验眼科杂志

Age-related macular degeneration (AMD) is a common cause of vision loss and living quality impairment in elderly people.Dry AMD is considered to be a neurodegenerative disease, and there has been no preventive method or effective therapy for it so far.Recent studies reveal that accumulation of lipofuscin may lead to dysfunction and even death of retinal pigment epithelium (RPE) cells.Autophagy is a major lysosome-dependent degradation pathway in eukaryotes involved in the disposal of damaged cytoplasmic proteins and organelles.Autophagy is revealed to be involved in the pathological processes of several neurodegenerative diseases such as Alzheimer's disease and dry AMD.Therefore,studies focus on autophagy may provide a new target for the prevention and treatment of dry AMD.This paper reviewed the research progress of autophagy in the pathogenesis of AMD in recent years.The roles of autophagy,lysosomal damage,oxidative stress and immune inflammatory reaction were described.

Effect of hypoxia on expressions of stromal cell-derived factor-1 and integrin-linked kinase in retinal pigment epithelium cell in vitro / 中华实验眼科杂志

Background Hypoxia is a crucial factor of neovascularization.Many researches found that stromal cell-derived factor-1 (SDF-1) and integrin-linked kinase (ILK) play an important role in the neovascular disease.However,effect of SDF-1 and ILK in eye neovascular disease is below understood.Objective The aim of this study was to investigate the effect of hypoxia on the expressions of SDF-1 and ILK in cultured retinal pigment epithelium(RPE) cells in vitro.Methods RPE tissue was isolated from 4-week-old C57BL/6 mouse and was digested and cultured in DMEM/F12 with 10％ fetal bovine serum (FBS).The cells with 80％ confluence were collected and passaged.The third generation of cells were identified with cytokeratin 18 (CK18) antibody by immunochemistry.The cells were inoculated at the density of 5×104 cells/ml to free-serum DMEM/F12 for 24 hours and then were cultured in regular medium in the normoxic control group.RPE cells were cultured for 1 hour and 3,6,12,24,48,72 hours with 200 μmol/L CoCl2 in the hypoxia group.Reverse transcription-PCR(RT-PCR) was used to evaluate the expressing change of SDF-1 mRNA and ILK mRNA in RPE cells,and Western blot was used to assay the expressing change of SDF-1 protein and ILK protein in RPE cells in different time points.The detected outcomes were represented as the ratio of target gene A value/β-actin A value.Results Cultured cells showed the polygon in shape with the black pigment granules in cytoplasm.Over 90％ cells were positive response for CK18.Expressions of the SDF-1 mRNA and ILK mRNA were increased in different time points after CoCl2 co-cultured(SDF-1 mRNA:F=281.875,P=0.000 ;ILK mRNA: F=187.566,P=0.000),with the highest expressing value in hypoxia at 12 hours.No significant change in the expression of SDF-1 mRNA and protein was found 1 hour after CoCl2 co-cultured,but expressions of SDF-1 mRNA and ILK mRNA were significantly higher in 3,6,12,24,48 and 72 hours than the normoxic control group(P＜0.01).The expressions of SDF-1 protein and ILK protein were gradually ascended with the time increase of CoCl2 co-culture,showing a significant difference among different time points(SDF-1: F=44.719,P =0.000 ; ILK: F =144.481,P =0.000),and the up-regulation of SDF-1 protein and ILK protein expression was seen mainly in 3,6,12,24,48 and 72 hours after CoCl2 co-cultured in comparison with the normoxic control group (P＜0.01).Conclusions SDF-1 and ILK are involved in the hypoxic response of RPE cells and may play a potential role in ischemic/hypoxic retinopathy.

Effects of amniotic membrane on proliferation and differentiation of human retinal pigment epithelial cell / 中华实验眼科杂志

Background Human retinal pigment epithelial (RPE) cell transplantation treating retinal degenerative diseases is a researching topic,and the source of human RPE cells is a key problem.Many biological carriers can be used for the preparation of RPE cell layer.However,some advantages,such as cytotoxicity,lack of stability and immunologic reaction etc.are still existed.To study an ideal biological carrier is very important.Objective This experimental was to determine the effects of amniotic membrane on the proliferation and differentiation of human RPE cells and the possibility as a scaffold for RPE cell transplantation.Methods ARPE19 cell line cells were cultured and passaged in DMEM/F12 medium with 10％ fetal bovine serum,and 8-12generation of cells were used.The cells were divided into two groups.One group of cells were incubated on the denuded amniotic membrane,and the other group of cells were cultured in the medium (control group).MTT was performed to detect the A492 value of RPE cells for the evaluation of cell proliferation ability 24,48,72,96 hours after culture.Cell morphology was compared by histopathological examination 3 weeks after culture.The mRNA expression of pigment epithelium-derived factor (PEDF),N-cadherin,β-catenin and cell connection related proteins in the cells of both groups were assayed using reverse transcription polymerase chain reaction (RT-PCR).Ultrastructure of the cells was observed under the transmission and scan electronic microscope 3 weeks after culture.Results The number of ARPE-19 cells cultured on denuded amniotic membrane was decreased significantly in comparison with the normal culture plate(F=41.760,P =0.000).Histopatholy also showed that the cell density on amniotic membrane was lower than of normal cells on plate surface.Moreover,the expression level of claudin 1 mRNA,N-cadherin mRNA and PEDF mRNA were significantly up-regulated in denuded amniotic membrane group in comparison with control group (t=15.828,P=0.000 ;t=6.839,P=0.002 ;t=14.667,P=0.000),but the expression of Connexin 43 mRNA was down-regulated in denuded amniotic membrane group compared with control group(t=3.358,P=0.024).Ultrastructural examination revealed that ARPE-19 cells cultured on amniotic membrane exhibited a polygonal epithelial phenotype with cilium on the apical side,however,the cells cultured on normal culture plate displayed fusiform shape and uneven thickness.Conclusions Amniotic membrane plays a promoting effect on the differentiation of ARPE-19 cells and a inhibitory effect on the proliferation of ARPE-19 cells,suggesting that amniotic membrane might be an useful scaffold for the preparation of functionally mature RPE cells for clinical transplantation.

Effect of hypoxia on expression of angiopoietin-2 in human retinal pigment epithelium / 中华实验眼科杂志

Background Choroidal neovascularization(CNV)has been descrihed as a main reason of visual loss in a lot of ocular diseases.Researches showed that local hypoxia and retinal pigment epithelial(RPE) cells play an important role in the formation of CNV.A closely relationship of angiopoietin-2 (Ang-2) and angiogenesis has been proved.However,whether the expression of Aog-2 in hypoxic cultured human RPE cells is associated with the pathogenesis of CNV is still below understood.Objective This study was to investigate the effects of hypoxia on expression of Ang-2 in cultured human RPE cells in vitro,and discuss the possible effects of Ang-2 in the formation of CNV.Methods Human RPE cells were cultured and passaged,and 4-7 generation of cells were used in the experiment.The cells were incubated in cultural plate at the density of 5×107 cells/L.The culture medium containing 200 μmol/L CoCl2 was used to establish the hypoxia model of human RPE cells culturecd in vitro for 0.5,1,2,4,6,12and 24 hours,and the RPE cells cultured under normoxia were as controls.Reverse transcription polymerase chain reaction(RT-PCR) was used to detect the expression of Ang-2 mRNA in cultured human RPE cells,and enzymelinked immunosorbnent assay(ELISA) was used to assay the content of Ang-2 protein in supernatant of cultured human RPE cells.Results The survival rate of human RPE cells was 90％ after resoscitation.The fourth generation of cells showed the fusiform with the less pigment in them.The Ang-2 mRNA/β-actin mRNA value in human RPE cells was significantly different among various groups(F=1086.30,P=0.00),The Ang-2 mRN A/β-actin mRNA value in hypoxia cultured for 0.5 hours group began to increase and peaked in hypoxia culture for 4-6 hours group,with the significant differences in comparison with normoxia control group(P＜0.05).The Ang-2 mRNA/β-actin mRNA value decreased to the baseline level at hypoxia for 24 hours.The ELISA analysis showed that the concentration of Ang-2 protein in supernatant of RPE cells showed significant difference among groups(F=1034.00,P=0.00).The concentration of Ang-2 protein increased at hypoxia culture for 0.5 hours and peaked at 6 hours,showing significant differences in comparison with the control group (P＜0.05).Conclusions Hypoxia could significantly up-regulate the expression of Ang-2 in human RPE cells cultured in vitro.Ang-2 expresses highly in the early stage of hypoxia,implying that Ang-2 participates in the formation of CNV.

Effects of basic fibroblast growth factor on growth phase of cultured bovine retinal pigment epithelium cells in vitro / 中国康复理论与实践

@#ObjectiveTo observe the effects of basic fibroblast growth factor (bFGF) on growth period of cultured bovine retinal pigment epithelium cells in different durations.MethodsDifferent proportion of phase with 4 ng/ml bFGF were measured by Flow Cytometry (FCM) assay.ResultsbFGF increased the RPE cells' proportion of phase S and decreased the proportion of phase G0-G1. ConclusionbFGF can stimulate RPE cells from phase G1 to phase S.

The changes of protein kinase C for human retinal pigment epithelium and retinal glial cells proliferation induced by the subretinal fluid / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To study the effect of the subretinal fluid (SRF) on proliferation of retinal pigment epithelium (RPE) cells and retinal glial (RG) cells and associated activation and translocation of protein kinase C (PKC) as well as the application of PKC inhibitor.MTEHODS: RPE and RG cells were disintegrated to obtain PKC activity of cytoplasm and cellular membrane after being treated by the subretinal fluid (SRF) from the different stages of PVR patients (grade B and C) or being treated with PKC specific activator [phorbol-12-myris-tate-13-acetate (PMA)] or normal vitreous or DMEM culture medium. PKC activity in cytoplasm and cellular membrane was measured using radioactive isotope 32P labeling in a specific reaction of phosphorylation on PKC substrate. In addition, the PKC inhibitor, dequalinium chloride, was used to pretreat the RPE and RG cells before the cells exposed to SRF or PMA or normal vitreous. 3H-TdR (tritiated thymidine) was used to measure the levels of proliferation of RPE and RG cells with or without the activation and translocation.RESULTS: SRF and PMA promoted the proliferation of RPE and RG cells. SRF and PMA activated PKC in the cytoplasm of RPE and RG cells and the activated cytoplasm PKC translocated to the cellular membrane of RPE or RG cells. The cell proliferation or PKC activation or translocation were not equally active in RPE as in RG cells. However, PKC inhibitor which attenuated the cell proliferation did not show significant difference on inhibition of RPE and RG cell proliferation. (P ＞0.05).CONCLUSION: SRF can lead to the activation and translocation of PKC in RPE and RG cells, which promote the proliferation of RPE and RG cells. Dequalinium chloride can inhibit PKC activation and translocation hence slow down the cells proliferation.

Inhibition of PCNA Antisense Oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells / 华中科技大学学报（医学）（英德文版）

The proliferating cell nuclear antigen (PCNA) gene expression was blocked and retinal pigment epithelium (RPE) proliferation was inhibited by using antisense oligonucleotides (AS-ODN)mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy (PVR). RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN (0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides (S-ODN with the same concentrations as AS-ODN) mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by 3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group.Expression of PCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density (AOD) was measured by image analysis system and was subjected to q-test and correlation analysis with CPM.Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN (0.28 and 1.12 μ mol/L) markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant (P＜0.05 and P＜0.01,repectively) as compared with blank control group. AOD was well correlated with CPM (r=0.975). It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

Inhibition of indomethacin on proliferation and DNA synthesis of cultured hRPE cells / 眼科研究

ObjectiveTo investigate the inhibitory effects of indomethacin(IN) on proliferation and DNA synthesis of cultured human fetal retinal pigment epithelium(hRPE) cells in vitro.MethodsPrimary culture and subculture of hRPE cells were established in vitro first.Cultured hRPE cells were treated by various concentrations 50,100,200,400,600 μ mol/L(final concentration)of IN for 24h.After 24h,the amount of DNA in RPE cells was determined by the absorbance at 280nm of Nucleic Acid δ Protein Analysis.Cells proliferation of RPE were measured with methyl thiazolyl tetrazolium(MTT) assay method by adding 100,200,400,600,800,1000μ mol/L(final concentration) of IN for 12h.ResultsAfter added various concentrations of IN,the DNA concentrations were ( 101.1712± 15.5124),( 88.6400± 13.5845),( 72.3651± 7.7969),( 59.9089± 10.7229),( 51.2236± 8.7757)μg/ml respectively,P values were 0.000,0.000,0.000,0.000,0.000(q test) as compared to that ( 213.7351± 83.1572)μg/ml in 0μg/L IN.The A values added 100,200,400,600,800,1000μmol/L of IN were ( 0.2367± 0.0546),( 0.1687± 0.0695),( 0.0819± 0.03461),( 0.0656± 0.01759),( 0.0554± 0.02865),( 0.0508± 0.02775)respectively,P values were 0 .158,0.000,0.000,0.000,0.000,0.000(q test) as compared to ( 0.2674± 0. 04302) of A value of 0ug/L IN.ConclusionThe data suggested that IN can inhibit DNA synthesis and proliferation of hRPE cells in vitro in a dose dependent manner.

Histologic Study in Transplantation of Cultured Rabbit Retinal Pigment Epithelium

The authors investigated the possibility of transplantion of cultured retinal pigment epithelial cell to normal pigmented rabbit retina. Focal retinal detachment was made in the pigment rabbit retina, and the cultured RPE cells were injected into subretinal space. The neural retina spontaneously rettached withim 6 days. At 4 weeks after tranplantation, eyes were enucleated and examined with light-microscopy and electron-microscopy. The transplanted RPE cells were proliferated with multilayer in electronmicroscopic finding, and the tight-junction was found between proliferative RPE cells. The outer segment and nucleus of photoreceptor cell were well preserved in microscopic finding. As a result, the cultured RPE cells can be sucessfully transplanted to normal pigmented rabbit retina, and photoreceptor cell was not changed after transplantation.