ObjectiveTo explore the effect of cathepsin L (CTSL) inhibitor on apoptosis of retinal pigment epithelial (RPE) cells and mitochondrial oxidative stress. MethodsRPE cells were cultured in vitro and divided into control group, hydrogen peroxide (H2O2) group, and H2O2+CTSL inhibitor group. The cells of H2O2 group and H2O2+CTSL inhibitor group were incubated in the medium containing 400 μmol/L H2O2 for 24 hours and 10 μmol/L CTSL inhibitor was added in H2O2+CTSL inhibitor group at the same time. The cells of normal group were routinely cultured cells. The follow-up experiment was carried out 24 hours after modeling. The rate of apoptosis was detected by flow cytometry. The expression of CTSL was detected by immunofluorescence staining, Western blot and real time-polymerase chain reaction. The level of mitochondrial super oxide was detected by MitoSOX fluorescent probe, and the mitochondrial structure was observed after MitoTracker staining, the average area, form factors, and branch of mitochondria were quantitatively analyzed. The two groups were compared using two-tailed Student t test, while numerous groups were compared using one-way ANOVA. ResultsCompared with control group, the rate of apoptosis in H2O2 group was significantly higher (t=3.307, P=0.029 7), the expression level of CTSL was significantly increased (t=19.950, 6.916, 14.220; P＜0.05). Compared with H2O2 group, the expression level of CTSL, the rate of apoptosis and the mitochondrial ROS level in H2O2+CTSL inhibitor group were significantly lower (t=11.940, 4.718, 16.680; P＜0.05). The mitochondria of H2O2+CTSL inhibitor group were elongated, oval-shaped or rod-shaped, while the mitochondria of H2O2 group lost their continuous contour shape and complete structure. The differences of the average area, form factors, and brach of mitochondria among 4 groups were statistically significant (F=251.700, 34.010, 60.500; P＜0.000 1). ConclusionsH2O2 can significantly induce apoptosis in RPE cells and increase CTSL expression. CTSL inhibitor can inhibit the H2O2-induced apoptosis of RPE cells, lower the mitochondrial super oxide level, and successfully repair the mitochondrial structure.
Objective To observe the effect of bone forming protein 4 (BMP4) on the proliferation and migration of human retinal pigment epithelium (RPE) cells under oxidative stress, and to preliminarily explore its effect on epithelial-mesenchymal transition (EMT) of RPE cells. MethodsHuman RPE cells cultured in vitro were divided into normal group, pure 4-hydroxynonenal (HNE) group (4-HNE group), 4-HNE+NC group and 4-HNE+ small interfering BMP (siBMP4) group. The effect of 4-HNE on the proliferation of RPE cells was detected by thiazole blue colorimetry. The effects of 4-HNE and BMP4 on cell migration were determined by cell scratch test. The expression of BMP4 was detected by immunofluorescence staining, Western blot and real-time quantitative polymerase chain reaction. The transfection efficiency of siBMP4 was observed by fluorescence microscopy. Mitochondrial reactive oxygen species (MitoSOX) were detected by flow cytometry. The expression of EMT markers E-cadherin and Fibronection were detected by immunofluorescence assay. t-test was used for comparison between the two groups, and one-way analysis of variance was used for comparison between the three groups. ResultsCompared with normal group, cell proliferation and migration ability of 4-HNE group were significantly enhanced, with statistical significance (t=21.619, 24.469; P＜0.05). The expression of BMP4 in cells was significantly increased, and the difference was statistically significant (t=19.441, P＜0.05). The relative expression levels of BMP4 mRNA and protein were also significantly increased, with statistical significance (t=26.163, 37.163; P＜0.05). After transfection with siBMP4 for 24 h, the transfection efficiency of BMP4 in RPE cells was＞90%. Compared with 4-HNE group and 4-HNE+NC group, the relative expression levels of BMP4 protein (F=27.241), mRNA (F=36.943), cell mobility (F=46.723) and MitoSOX expression levels (F=39.721) in normal group and 4-HNE+siBMP4 group were significantly decreased. The differences were statistically significant (P＜0.05). The epithelial marker E-cadherin increased significantly, while the mesenchymal marker Fibronection decreased significantly, with statistical significance (F= 51.722, 45.153; P＜0.05). ConclusionsBMP4 inhibits RPE proliferation and migration under oxidative stress. BMP4 is involved in inducing EMT in RPE cells.
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 explore the effect of bone morphogenetic protein 4 (BMP4) on the glycolysis level of human retinal microvascular endothelial cells (hRMECs). MethodsA experimental study. hRMECs cultured in vitro were divided into normal group, 4-hydroxynonenal (HNE) group (4-HNE group) and 4-HNE+BMP4 treatment group (BMP4 group). 4-HNE group cell culture medium was added with 10 μmmol/L 4-HNE; BMP4 group cell culture medium was added with recombinant human BMP4 100 ng/ml after 6 h stimulation with 10 μmol/L 4-HNE. The levels of intracellular reactive oxygen species (ROS) were detected by flow cytometry. The effect of 4-HNE on the viability of cells was detected by thiazole blue colorimetry. Cell scratch test and Transwell cell method were used to determine the effect of 4-HNE on cell migration. The relative expression of BMP4 and SMAD9 mRNA and protein in normal group and 4-HNE group were detected by real-time quantitative polymerase chain reaction and Western blot. Seahorse XFe96 cell energy metabolism analyzer was used to determine the level of intracellular glycolysis metabolism in normal group, 4-HNE group and BMP4 group. One-way analysis of variance was used for comparison between groups. ResultsThe ROS levels in hRMECs of normal group, 4-HNE group and BMP4 group were 21±1, 815±5, 810±7, respectively. Compared with the normal group, the levels of ROS in the 4-HNE group and the BMP4 group were significantly increased, and the difference was statistically significant (F=53.40, 50.30; P＜0.001). The cell viability in the normal group and 4-HNE group was 1.05±0.05 and 1.28±0.05, respectively; the migration rates were (0.148±0.005)%, (0.376±0.015)%; the number of cells passing through the pores were 109.0±9.6, 318.0±6.4, respectively. Compared with the normal group, the 4-HNE group had significantly higher cell viability, cell migration rate, and the number of cells passing through the pores, and the differences were statistically significant (F=54.35, 52.84, 84.35; P＜0.05). The relative expression levels of BMP4 and SMAD9 mRNA in the cells of the 4-HEN group were 1.680±0.039 and 1.760±0.011, respectively; compared with the normal group, the difference was statistically significant (F=53.66, 83.54; P＜0.05). The relative expression levels of BMP4 and SMAD9 proteins in the cells of the normal group and 4-HEN group were 0.620±0.045, 0.860±0.190, 0.166±0.049, 0.309±0.038, respectively; compared with the normal group, the differences were statistically significant (F=24.87, 53.84; P＜0.05). The levels of intracellular glycolysis, glycolytic capacity and glycolytic reserve in normal group, 4-HNE group and BMP4 group were 1.21±0.12, 2.84±0.24, 1.78±0.36, 2.59±0.11, 5.34±0.32, 2.78±0.45 and 2.64±0.13, 5.20±0.28, 2.66±0.33. Compared with the normal group, the differences were statistically significant (4-HNE group: F=86.34, 69.75, 58.45; P＜0.001; BMP4 group: F=56.87, 59.35, 58.35; P＜0.05). There was no significant difference in intracellular glycolysis, glycolysis capacity and glycolysis reserve level between 4-HNE group and BMP4 group (F=48.32, 56.33, 55.01; P＞0.05). ConclusionBMP4 induces the proliferation and migration of hRMECs through glycolysis.
With the advancement of molecular biology technology and the development of genetics, the viral vector system has been continuously improved and optimized. The viral vector system has gradually become one of the best carriers in ophthalmic gene therapy. Adenovirus vector has the characteristics of transient expression and plays an important role in reducing corneal immune response. Lentiviral vector has the characteristics of stable and high efficiency and can be expressed slowly in the body for a long time.Adeno-associated virus vector has the characteristics of low immunogenicity, high efficiency and precision and can infect a variety of retinal cells. The combined use of adeno-associated virus vector and CRISPR-Cas9 provides new methods for precise treatment of ophthalmic genetic diseases. The advent of viral vectors has significantly increased the potential of gene therapy and has unparalleled advantages over traditional therapies. We have reason to believe that virus-based gene transduction technology will soon achieve clinical application in the near future, and a large number of difficult ophthalmic problems will be solved by then.
With the development of life sciences and informatics, bioinformatics is developing as an interdisciplinary subject. Its main application is the relationship between genes and proteins and their expression. With the help of genomics, proteomics, transcriptomics, and metabolomics, researchers introduce bioinformatics research methods into fundus disease research. A series of gratifying research results have been achieved including the screening of genetic susceptibility genes, the screening of diagnostic markers, and the exploration of pathogenesis. Genomics has the characteristics of high efficiency and accuracy. It has been used to detect new mutation sites in retinoblastoma and retinal pigment degeneration research, which helps to further improve the pathogenesis of retinal genetic diseases. Transcriptomics, proteomics, and metabolomics have high throughput characteristics. They are used to analyze changes in the expression profiles of RNA, proteins, and metabolites in intraocular fluid or isolated cells in disease states, which help to screen biomarkers and further elucidate the pathogenesis. With the advancement of technology, bioinformatics will provide new ideas for the study of ocular fundus diseases.
ObjectiveTo investigate the protective effect of butylphenyphthalein (NBP) on RPE apoptosis induced by H2O2.MethodsThe human RPE cell line (human ARPE-19 cell line) were used as the experimental cells and were divided as control group, model group, NBP group. Complete medium was used in control group. The model group was stimulated with 200 μmol/L H2O2 for 2 h, and the cells were cultured in complete medium. The NBP group was cultured with 200 μmol/L H2O2 and 1 μmol/L NBP for 2 h. After changing the medium, complete medium was combined with 1 μmol/L NBP to continue the culture of the cells. Cell viability were detected by MTT assay while the morphology of RPE were observed by HE staining. Moreover, Hoechst 33258 was used to detect RPE cell apoptosis. Mitochondrial membrane potential (JC-1) staining were performed to monitor changes in cell membrane potential and the characteristic change of apoptosis in RPE cells. Furthermore, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining were used to analyze the effect of NBP treatment on the expression of ROS. The effect of NBP on the expression of Heme oxygenase-1(HO-1) was analyzed by cellular immunofluorescence and western blotting.ResultsThe results of MTT assay showed that the cells were cultured for 24 and 48 hours, cell viability of control group (t=17.710, 13.760; P＜0.000 1, ＜0.000 1) and treatment group (t=4.857, 9.225; P=0.000 7, ＜0.000 1) were stronger than that of model group, and the difference was statistically significant. HE staining and Hoechst33258 staining showed that compared with the control group, the number of cells in the model group was significantly less, and the cell morphology was incomplete. Compared with the model group, the number of cells in the treatment group was significantly increased, and the cell morphology was better. The results of JC-1 assay showed that the number of apoptotic cells in the model group was significantly higher than that in the control group, and the number of apoptotic cells in the treatment group was significantly lower than that in the model group. DCFH-DA staining showed that the ROS accumulation in the model group was more than that in the control group, and the ROS accumulation in the treatment group was less than that in the model group. Immunostaining observation showed that the HO-1 fluorescence intensity of the cells in the treatment group was significantly higher than that of the control group, and the difference was statistically significant (t=10.270, P=0.000 5). Western blot analysis showed that NBP up-regulated the expression level of HO-1 in a time-dependent manner. The relative expression of HO-1 at 4, 8, and 12 h of NBP showed a clear increase trend compared with 0 h, and the difference was statistically significant (F=164.91, P＜0.05).ConclusionsOxidative stress injury can down-regulate the viability of RPE cells and induce apoptosis. NBP can increase the antioxidant capacity of RPE cells, reduce cell damage and inhibit cell apoptosis by up-regulating HO-1 expression.
ObjectiveTo observe the effects of NDRG1 on proliferation, migration and lumen formation of retinal vascular endothelial cells (RF/6A cells) in monkeys under high glucose condition. MethodsRF/6A cells were divided into normal group, mannitol group, high glucose group, small interfering RNA (siRNA) negative control group without target gene (siRNA group), 30 nmol/L siRNA down-regulated NDRG1 genome (siNDRG1 group) and 50 nmol/L siNDRG1 group. Normal group cells were cultured conventionally. The mannitol group was added with 25 mmol/L mannitol, and the high-glucose group was added with 25 mmol/L glucose. In the siRNA group, 25 mmol/L glucose was added, and then blank siRNA was added for induction. The 30 and 50 nmol/L siNDRG1 groups were added with 25 mmol/L glucose and induced with 30 and 50 nmol/L siRNDRG1, respectively. All cells were incubated for 24 h for follow-up experiments. Cell proliferation was observed by 4', 6-diaminidine 2-phenylindole staining. Cell counting kit-8 staining was used to detect cell activity. The expression level of NDRG1 mRNA and protein was detected by Western blot and real-time quantitative polymerase chain reaction. Cell migration was observed by cell scratch assay. Cell lumen formation assay was used to detect lumen formation. The two-tailed Student t test was used to compare the two groups. One-way analysis of variance was used to compare groups. ResultsThere were significant differences in cell proliferation rate (t=36.659, 57.645) mobility rate (t=24.745, 33.638) and lumen formation number (t=41.276, 22.867) between high glucose group and normal group and mannitol group (P＜0.01). Compared with normal group and mannitol group, the relative expression levels of NDRG1gene mRNA and protein in high glucose group were significantly decreased, with statistical significance (t=46.145, 21.541, 36.738, 32.976; P＜0.001). Compared with the siRNA negative group, the relative expression levels of NDRG1gene mRNA and protein in 30 nmol/L siNDRG1 group and 50 nmol/L siNDRG1 group were significantly decreased, and the differences were statistically significant (t=44.275, 40.7577, 57.167, 25.877; P＜0.01). Compared with normal group and siRNA group, cell mobility in 30 nmol/LsiNDRG1 group was increased, and the difference was statistically significant (t=57.562, 49.522; P＜0.01). Compared with normal group and siRNA group, the number of cell lumen formation in 30 nmol/LsiNDRG1 group was significantly increased in the same field of vision, and the difference was statistically significant (t=63.446, 42.742; P＜0.01). ConclusionDown-regulation of NDRG1 gene can improve the activity, migration and lumen formation of RF/6A cells under hyperglycemia.
Objective To observe the expression of miR-204 and 211 human embryonic stem cells (hESCs) differentiated into retinal pigment epithelial (RPE) cells. Methods RPE cells were derived from hESCs by natural differentiation method, and were identified. miRNA expression profiles and real-time polymerase chain reaction (RT-PCR) of miR-204 and 211 were generated from the following groups: hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and human fetal RPE (hfRPE) cells. Results miRNA-204 was continuously upregulated throughout the entire differentiation process of hESCs to RPE cells. It increased 5.026 times in hESCs-derived cells containing pigmented foci compared to hfRPE cells; it was increased 3.337 times in hESCs-derived RPE cells compared to hESCs-derived cells containing pigmented foci; it increased 13.574 times in hfRPE cells compared to hESCs-derived RPE cells. miR-211 does not change during differentiation from hESC to RPE, but it increased 44.333 times in hESCderived RPE cells compared to hfRPE cells. miR-211 was the biggest difference in the miRNA expression pattern. In four cell types of hESCs, hESCs-derived cells containing pigmented foci, hESCs-derived RPE cells and hfRPE cells, RT-PCR showed the levels of miR-204 were 91.81plusmn;4.43, 2263.09plusmn;206.39, 5996.80plusmn;235.42, and 171676.45plusmn;999.82 respectively. miR-204 was significantly increased during the whole course (t=18.22, 20.66, 279.38;P＜0.001). The levels of miR-211 were 2.23plusmn;0.31, 129.33plusmn;3.75, 125.7592plusmn;4.78, and 16682.00plusmn;352.97 respectively. miR-211 was significantly increased from hESCs to cells containing pigmented foci and from hESCs-derived RPE cells to hfRPE (t=58.58, 81.24; P＜0.001). Conclusion There is a continuous change of miR-204 and 211 in differentiation of RPE cells from hESCs.