Objective To observe the expression of proteins in light-injured retinal pigment epithelial (RPE) cells. Methods ARPE19 cells were exposed to the cool white light at the intensity of (2200plusmn;300) Lx for 6 hours to set up the light injured model. Cellular soluble proteins was extracted and analyzed by means of twodimensional electrophoresis to find out the changes of protein map of lightinjured RPE cells. Results Cellular soluble proteins had (390plusmn;10) spots on the map, in which 11 spots had obvious difference between the light injured group and the normal control group. In the lightinjured cells, the expressio of 8 proteins increased, 1 decreased, and 2 disappeared. Conclusion Twodimensional electrophoresis can find out the difference of expression of proteins in lightinjured and normal RPE cells.
ObjectiveTo preliminarily investigate the mechanism of MMP-9 blocking CD73 detachment from RPE cells surface and preventing and treating experimental autoimmune pigment membranitis (EAU).MethodsRPE cells isolated from wild-type C57BL/6 and CD73 gene knockout (CD73-/-) mice were cultured in vitro, and treated with lipopolysaccharide and TNF-α to induce CD73 detachment from RPE surface. According to whether MMP-9 inhibitor CTK8G1150 was added at the same time (the final concentration was 5.0 mol/L) or not, RPE cells cultured in the two types of mice were respectively set as MMP-9 inhibitor intervention group and non-intervention control group. The cells in each group were treated with the intervention of a solvent, 1 μmol/L adenosine monophosphate (AMP), 1 μmol/L AMP, and 3 μmol/L 5' -α,β-methylene adenosine diphosphate (APCP) (AMP+APCP). The stimulating effect of RPE cells in different groups on CD4+ T cell proliferation was detected by tritiated thymidine incorporation. Adoptive immune induced EAU in wild-type B6 mice and CD73-/- mice, respectively. The receptor mice were randomly divided into the MMP-9 inhibitor intervention group and the non-intervention control group, and CTK8G1150 or the solvent were injected into the subretinal cavity 4, 7 and 10 days after adoptive immunity. CD73 mRNA and protein expression in RPE cells of recipient mice were detected by real-time quantitative PCR (RT-PCR) and Western blot. One-way ANOVA was used to analyze all experimental data.ResultsWhen the stimulation mode was AMP, the proliferation of CD4+ T cells in the C57BL/6 MMP-9 inhibitor intervention group decreased significantly compared with the non-intervention group (F=13.28, P＜0.01). When the stimulation mode was solvent and AMP+APCP, there was no statistically significant difference in the proliferation capacity of CD4+ T cells between the two groups (F=7.78, 6.58; P＞0.05). There was no statistically significant difference in the proliferation capacity of CD4+ T cells between the CD73-/- MMP-9 inhibitor intervention group and the non-intervention group (F=5.24, 6.12, 7.04; P＞0.05). RT-PCR results showed that there was no statistically significant difference in the relative expression of CD73 mRNA in RPE cells between the MMP-9 inhibitor group and the non-intervention control group (F=6.54, P>0.05). Western blot results showed that the expression of CD73 protein in RPE cells in the MMP-9 inhibitor group of B6 receptor mice was significantly increased compared with the control group (F=15.24, P＜0.01).ConclusionMMP-9 inhibitor blocks CD73 detachment from RPE cells surface and has a protective effect on EAU.
Objective To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L nor-Arginine (nor-NOHA) on high glucose cultured rhesus macaque retinal vascular endothelial cell line (RF/6A) in vitro. Methods The RF/6A cells were divided into the following 4 groups: normal control group (5.0 mmol/L of glucose, group A), high glucose group (25.0 mmol/L, group B), high glucose with 125 mg/L nor-NOHA group (group C), and high glucose with 1% DMSO group (group D). The proliferation, migration ability and angiogenic ability of RF/6A cells were measured by Methyl thiazolyl tetrazolium (MTT), transwell chamber and tube assay respectively. The express of Arg I, eNOS, iNOS mRNA of RF/6A cells were measured by real-time polymerase chain reaction (RT-PCR), Enzyme-linked immuno sorbent assay (ELISA) was used to detect the expression of NO and interleukine (IL)-1b of RF/6A cells. Results The proliferation, migration, and tube formation ability of group A (t=2.367, 5.633, 7.045;P＜0.05) and group C (t=5.260, 6.952, 8.875;P＜0.05) were significantly higher than group B. RT-PCR results showed the Arg I and iNOS expression in group B was higher than that in group A (t=6.836, 3.342;P＜0.05) and group C (t=4.904, 7.192;P＜0.05). The eNOS expression in group B was lower than that in group A and group C (t=4.165, 6.594;P＜0.05). ELISA results showed NO expression in group B was lower than that in group A and group C (t=4.925, 5.368;P＜0.05). IL-1b expression in group B was higher than that in group A and group C (t=5.032, 7.792;P＜0.05). Conclusions Nor-NOHA has a protective effect on cultured RF/6A cells in vitro and can enhance its proliferation, migration and tube formation. The mechanism may be inhibiting the oxidative stress by balancing the expression of Arg/NOS.
Objective To build the lentiviral vectors of pigment epithelial derived factor (PEDF) gene, and investigate their expression in human umbilical cord mesenchymal stem cells (hUCMSCs). Methods The PEDF lentiviral vectors (LV-PEDF) were built by DNA recombination and confirmed by DNA sequencing. hUCMSCs were transfected by LV-PEDF with MOI 10, 30, 50, respectively. The transfection efficiency was observed under fluorescence microscope. Cell immunofluorescence, immunocytochemistry and real-time PCR methods were used for detecting the expression of PEDF and VEGF. Results The PEDF cDNA was sub-cloned into pCDH-CMV-MCS-EF1-copGFP vector successfully. DNA sequencing analysis confirmed that PEDF gene sequence was exactly the same with that reported in GenBank. pCDH-PEDF infected cells could show green fluorescence under fluorescence microscope. The transfection efficiency was 72.1% in PEDF-MSCs. Immunofluorescence and immunochemical staining confirmed that PEDF protein was overexpressed in hUCMSCs. The relative expression of PEDF mRNA in experimental group and control group was (0.170±0.028) and (0.015±0.007) respectively by RT-PCR, the difference was statistically significant (P＜0.001). The relative expression levels of VEGF mRNA in the two groups were (0.265±0.022) and (0.285±0.049), respectively, with no significant difference (P＞0.05). Conclusions We successfully built a lentivirus vector carrying PEDF gene and obtained hUCMSCs with overexpressed PEDF.
Objective To investigate the effects of epidermal growth factor (EGF)，fibroblast growth factor(FGF), and bovine serum on proliferation and apoptosis of the cultured fetal human retinal cells.Methods EGF and FGF were added or not to the medium of fetal human retinal cells cultured by bovine serum in vitro. The number of cells, bromodeoxyuridine(BrdU) incorporation and Tdt-mediated dUTP nick end labelling(TUNEL) were detected to determine the proliferation and apoptosis. Immunohistochemical staining of neuron specific enolase(NSE), Thy1.1, glial fibrillary acidic protein(GFAP) and scan electromicroscopy were performed to identify cell components. The expression of transcription factor c-fos, c-jun and apoptosis regulation factor bcl-2 and Bax were examined by immunohistochemical staining to explore the underlying mechanism.Results The increased number of NSE and Thy1.1 positive cells and BrdU incorporation, and decreased apoptotic cells were found in the groups treated with EGF and FGF. Meanwhile, the up-regulation of c-fos, c-jun and bcl-2 were also found. Conclusion EGF and FGF can promote the survival and proliferation of cultured retinal cells by up-regulating the expression of c-fos, c-jun and bcl-2. (Chin J Ocul Fundus Dis，2003，19：113-116)
ObjectiveTo observe the MiSeq sequencing analysis results of fulvic acid (FA) intervention in hypoxia-induced human retinal microvascular endothelial cell (hRMEC) gene expression profile.MethodshRMEC were cultured in vitro and divided into the hypoxia group (hypoxia treatment) and the FA intervention group (FA intervention after hypoxia). The MTT colorimetric method was used to detect the influence of different concentrations and different modes of FA on hRMEC activity. The optimal concentration of FA was chosen. RT-PCR was used to investigated the effect of FA on hypoxia-induced intercellular adhesion molecule-1 (ICAM-1), IL-1β, IL-4, IL-6, IL-6, IL-8, IL-10, MMP-2, TNF-α, TNF-β, other inflammatory factors in hRMEC, and inflammation-related factors mRNA expression. Cells in the hypoxia group and FA intervention group in the logarithmic growth phase were collected. MiSeq sequencing technology was applyed to complete the whole transcriptome sequencing of the two groups of cells, biological data were obtained, and the differentially expressed miRNA were analyzed on this basis. Gene annotation (GO) functionally significant enrichment analysis and Kyoto Encyclopedia of Genes and Genome (KEGG) pathway significant enrichment analysis were used to analyze the functions and signal pathways of differential miRNAs. The expression of inflammatory factors and inflammation-related factors were compared between groups. The expression level of the corresponding miRNA in the cell was regulated by miRNA mimic, and its effect on cell function was observed, so as to judge the effect of the miRNA.ResultsDifferent concentrations and different modes of action of FA had no effect on the cell viability of hRMEC. The mRNA expression of ICAM-1, IL-1β, IL-6 and TNF-β in the hypoxia group hRMEC were significantly up-regulated compared with the normal group, and the difference was statistically significant (t=3.426, 6.011, 5.282, 6.500; P=0.027, 0.004, 0.006, 0.003); the mRNA expression of ICAM-1, IL-6, TNF-α and TNF-β in the FA intervention group hRMEC was significantly lower than that of the hypoxia group, and the difference was statistically significant (t=9.961, 3.676, 3.613, 3.387; P=0.001, 0.021, 0.023, 0.028). There were 14 differentially expressed miRNAs between the hypoxia group and the FA intervention group, of which 9 were up-regulated genes and 5 were down-regulated genes. The predicted target genes of 4 differential miRNAs (hsa-miR-1285-3p, hsa-miR-30d-3p, hsa-miR-3170, hsa-miR-7976) were all ICAM-1. The results of significant enrichment analysis of GO function showed that the functions of differential genes were mainly enriched in the process of cell development, cell differentiation and single organism development. Significant enrichment analysis of the KEGG pathway showed that the differential miRNA expression was highly enriched in the proteoglycan pathway and the cytokine-cytokine receptor interaction pathway in cancer, and the arachidonic acid metabolism pathway and the amphetamine pathway were the more obvious differential expressions.ConclusionFA may affect the expression level of downstream ICAM-1 mRNA by regulating the expression of multiple miRNAs, thereby affecting the inflammatory state of cells after hypoxia-stimulated hRMEC.
Objective To observe biological characteristics of microencapsulated human endostatin/293 (hES/293) cells at different density and their inhibitory effects on the proliferation of human umbilical vein endothelial cells (HUVEC). Methods The microencapsulated hES/293 cells at different cellular density of 1×104 (group A), 1×106 (group B) and 1×108 (group C) cells/ml were made by polyelectrolyte complexometry technology. The empty microcapsules were set as control group (group D). Each group has 6 samples. After 1, 3, 7, 14 and 35 days in culture, the number of total cells, viable cells was counted by trypan blue staining, and the survival fraction was measured. The grow status of hES/293 cells was measured by MTT assay, and the concentration of endostatin protein in supernatant was measured by enzyme linked immunosorbent assay (ELISA). HUVECs were cocultured with hES/293 cells of group A, B and C. The proliferation of HUVEC at the 24, 72 and 120 hours after coculture was measured by MTT assay. Results The number of total cells and viable cells were increasing and the survival fraction reached its peak after 3 days in culture in group A, B and C. The growth rate in group A was higher than that in group B and C after 3 days in culture (P＜0.05), but the growth rate in group B was higher than that in group A and C after 7, 14 and 35 days in culture (P＜0.05). The concentration of endostatin protein in the supernatant was the same in group A, B and C after 1 and 14 days in culture (P＞0.05). However, group A had higher endostatin than group B and C after 3 days in culture, group B had higher endostatin higher than group A and C after 7 and 35 days in culture (P＜0.05). The hES/293 cells of group A, B and C had no effects on the proliferation of HUVEC(P＞0.05) after 24 hours coculture, but can inhibit the proliferation of HUVEC after 72 or 120 hours co-culture (P＜0.05). Conclusions The microencapsulated hES/293 cells at a density of 1×106 cells/ml can grow and survive, and release endostatin protein stably. The microencapsulated hES/293 cells at different density all can inhibit the proliferation of HUVEC.
ObjectiveTo observe the effect of subretinal injection of retinal pigment epithelium (RPE) cells for RPE in mice. MethodsA total of 30 postnatal day 7 C57BL/6J mice were randomly divided into normal mice group, OIR model group and OIR model cell transplanted group, 10 mice in each group. The OIR model was induced in mice of OIR model group and OIR model cell transplanted group. The RPE cells were subretinal injected into the RPE of mice in OIR model cell transplanted group. At 20 days after the injection, the RPE thickness was evaluated by fluorescence microscope. The expression of RPE65, Bestrophin and zonula occludens-1 (ZO-1) were estimated by Western blot and real-time quantitative PCR (RT-PCR). ResultsThe thickness of RPE in OIR model mice was thinner than that in normal mice; the thickness of RPE in OIR model cell transplantation mice was significantly thicker than that in the OIR model mice. The results of Western blot and RT-PCR indicated that the differences of protein (F=8.597, 18.864, 25.691) and mRNA expression (F=39.458, 11.461, 34.796) of RPE65, Bestrophin, ZO-1 were statistically significant between OIR model group and OIR model cell transplanted group (P < 0.05). ConclusionsSubretinal injection of RPE cells can promote RPE thickening. RPE65 and Bestrophin protein relative expression levels increased, ZO-1 protein relative expression levels reduced; mRNA expression levels of RPE65, Bestrophin and ZO-1 genes increased.
ObjectiveTo observe the effect of pyrimidine bundle-binding protein-associated splicing factors (PSF) on the function of hypoxia-induced human retinal microvascular endothelial cells (hRMECs).MethodsA three-plasmid system was used to construct lentivirus (LV)-PSF. After LV-PSF infected hRMECs in vitro, the infection efficiency was measured by flow cytometry. Real-time quantitative PCR (RT-PCR) was used to detect the expression of PSF mRNA in hRMECs infected with LV-PSF. The experiment was divided into two parts, in vivo and in vitro. In vivo experiments: 20 healthy C57B/L6 mice at the age of postnatal 7 were randomly divided into normal group, oxygen-induced retinopathy (OIR) group, OIR+LV-Vec group, and OIR+LV-PSF group, each group has five mice. Mice in 3 groups were constructed with OIR models except the normal group and the mice in OIR group were not treated. The mice in the OIR + LV-Vec group and the OIR+LV-PSF group were injected with an empty vector (LV-Vec) or LV-PSF in the vitreous cavity, respectively. The effect of LV-PSF on the formation of retinal neovascularization (RNV) was observed then. In vitro experiments: hRMECs were divided into normal group, hypoxia group, vector group, and PSF high expression group. HRMECs in the normal group were cultured in vitro; hRMECs in the hypoxic group were restored to normal culture conditions for 3 h after 3 h of hypoxia stimulation; hRMECs in the vector group and PSF high expression group were infected with LV-Vec and LV-PSF for 48 h, and hRMECs were returned to normal culture conditions for 24 h with hypoxia stimulation for 3 h. The effect of PSF on cell proliferation was observed by MTT colorimetry. Cell scratch test and Transwell migration experiment were used to observe the effect of PSF on cell migration ability under hypoxia stimulation. RT-PCR was used to observe the mRNA expression of HIF-1α, VEGF and PSF in each group of cells.ResultsThe LV-PSF of stably expressing PSF was successfully constructed. The infection efficiency was 97% determined by flow cytometry. The level of PSF mRNA in hRMECs infected with LV-PSF was significantly increased and detected by RT-PCR. In vivo experiments: The RNV area of the mice in the OIR group and the OIR + LV-Vec group was significantly increased compared to the normal group (t=18.31, 43.71), and the RNV area of the mice in the OIR + LV-PSF group was smaller than that in the OIR group (t=11.30) and OIR + The LV-Vec group (t=15.47), and the differences were statistically significant (P＜0.05). In vitro experiments: MTT colorimetry results showed that the proliferative capacity of hRMECs in the hypoxic group was significantly enhanced compared with the normal group (t=2.57), and the proliferative capacity of hRMECs in the PSF high expression group was significantly lower than that of the normal, hypoxic, and vector groups (t=5.26, 5.46, 3.73), the differences were statistically significant (P＜0.05). The results of cell scratch test showed that the hRMECs could be stimulated by the hypoxia stimulation for 3 hours to restore the normal condition for 24 hours or 48 hours (t=8.35, 13.84; P＜0.05). Compared with the vector group, cell migration rate in the PSF-high expression group was not significant (t=10.99, 18.27, 9.75, 8.93, 26.94, 7.01; P＜0.05). Transwell experiments showed that the number of cells stained on the microporous membrane was higher in the normal group and the vector groups, while the number of cells stained in the PSF high expression group was significantly reduced (t=9.33, 6.15; P＜0.05). The results of RT-PCR showed that the mRNA expression of HIF-1α and VEGF in hRMECs in the hypoxic and vector groups increased significantly compared with the normal group (t=15.23, 21.09; P＜0.05), but no change in the mRNA expression of PSF (t=0.12, 2.15; P>0.05); compared with the hypoxia group and the vector group, the HIF-1α and VEGF mRNA expression in hRMECs in the PSF high expression group were significantly decreased (t=10.18, 13.10; P＜0.05), but the PSF mRNA expression increased (t=65.00, 85.79; P<0.05).ConclusionPSF can reduce the RNV area in OIR model mice. PSF may inhibit hypoxia-induced proliferation and migration of hRMECs through the HIF-1α/VEGF signaling pathway.