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.
Objective To observe the effect of tetramethypyrazine (TMP) on the expression of hypoxia-related factors in human umbilical vein endothelial cells (HUVECs). Methods The second to fifth passage cultured HUVECs were divided into five groups: control group, CoCl2induced hypoxic group and 50, 100, 200 mu;mol/L TMP treatment groups. HUVECs in control group were not treated. HUVECs inCoCl2induced hypoxic group were treated with 150 mu;mol/LCoCl2for four hours. HUVECs in 50, 100, 200 mu;mol/L TMP treated groups were pretreated with 150 mu;mol/LCoCl2 for four hours, followed by treatment with 50, 100, 200 mu;mol/L TMP for eight hours. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of prolyl hydroxylase 2 (PHD2), hypoxia-induced factor-1alpha;(HIF-1alpha;) and vascular endothelial growth factor (VEGF). Protein levels of PHD2, HIF-1alpha;, and VEGF were detected using Western blot. Results Compared with the control group, theCoCl2 induced hypoxic group showed decreased mRNA and protein levels of PHD2 (t=3.734, 3.122;P＜0.05), while those of HIF-1alpha; and VEGF increased (HIF-1alpha; mRNA:t=4.589,P＜0.05; HIF-1alpha; protein:t=3.778,P＜0.05. VEGF mRNA:t=3.926,P＜0.05; VEGF protein:t=3.257,P＜0.05). Compared with theCoCl2 induced hypoxic group, 50, 100, 200 mu;mol/L TMP treated groups showed increased mRNA and protein levels of PHD2 (PHD2 mRNA: t=3.286, 3.617, 3.886;P＜0.05. PHD2 protein: t=2.813, 3.026, 3.078; P＜0.05); while those of VEGF decreased (VEGF mRNA: 50 mu;mol/L TMP: t=1.696,P＞0.05; 100 mu;mol/L TMP:t=2.974,P＜0.05; 200 mu;mol/L TMP: t=3.492,P＜0.05; VEGF protein: 50 mu;mol/L TMP: t=1.986,P＞0.05; 100 mu;mol/L TMP: t=2.976,P＜0.05; 200 mu;mol/L TMP:t=3.136,P＜0.05); although changes in HIF-1alpha;mRNA levels were not statistically significant (t=1.025, 0.726, -1.386;P＞0.05), showed a decrease in HIF-1alpha;protein levels (50 mu;mol/L TMP: t=2.056,P＞0.05; 100 mu;mol/L TMP:t=3.058,P＜0.05; 200 mu;mol/L TMP:t=3.828,P＜0.05). ConclusionIn HUVECs, TMP can upregulate the mRNA and protein expression of PHD2, while down regulating HIF-1alpha; protein expression and VEGF mRNA and protein expression under acute hypoxic conditions.
Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell (RGC). Methods Four short hairpin (sh) RNA interference sequences targeting rat Sirt1 gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction (PCR) and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirt1-1, si-Sirt1-2, si-Sirt1-3, si-Sirt1-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirt1 mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8×108 TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirt1 mRNA and protein were significantly decreased in the si-Sirt1-1, si-Sirt1-2, si-Sirt1-3 and si-Sirt1-4 groups (F=27.682, 1 185.206; P=0.000, 0.000). The si-Sirt1-2 group had the strongest effect in reducing the expression of Sirt1 mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirt1 gene can effectively down regulate the expression of Sirt1 mRNA and protein in RGC cells.
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 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.
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)
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 investigate the protection and the corresponding molecular mechanisms of polypyramidine tract binding protein-associated splicing factor (PSF) overexpression on human retinal microvascular endothelial cells (hRMECs) induced by advanced glycation end-products (AGEs).MethodsThe hRMECs were divided into the normal group, the vector group, PSF group, zinc protoporphyrin (ZnPP) group and PSF+ZnPP group for experiment. Cells in the normal group were cultured in a DMEM medium containing 10% fetal calf serum, penicillin/streptomycin, and placed in a closed constant temperature incubator at 37 °C, 95% air, and 5% CO2. Cells in the vector group were infected with empty lentivirus. The cells in the PSF group were infected with overexpressing PSF lentivirus. Cells in the ZnPP group were treated with ZnPP (10 mol/L) for 2 h. The PSF+ZnPP group cells were infected with overexpressing PSF lentivirus, and then pretreated with ZnPP (10 mol/L) for 2 h. With the last four groups of cells stimulated with AGEs, HE, Hoechst33258 staining and flow cytometry were used to observe the protective effect of high expression of PSF on cell damage and the antagonistic effect of ZnPP on PSF. Western blot was used to detect the protein expression of heme oxygenase-1 (HO-1), phosphorylated (p) extracellular regulatory protein kinase (ERK), and Nrf2 in the cells. U0126, a specific antagonist of ERK pathway, was introduced, and Western blot verified the reversal effect of U0126 on the expression of HO-1 induced by PSF protein.ResultsHE staining and Hoechst33258 staining showed that the number of nuclei of damaged cells of PSF group were significantly increased compared with control group, while decreased compared with PSF+ZnPP group (F=27.5, 38.7; P＜0.05). The results of flow cytometry showed that the ROS produced by cells in the PSF group was significantly increased compared to the normal group, and significantly decreased compared to the PSF+ZnPP group, the difference was statistically significant (F=126.4, P＜0.05). Western blot results showed that HO-1 expression of PSF group was significantly increased compared with control and the vector group (F=70.1, P＜0.05). AGEs inducement of 30, 60, 120 and 240 min could significantly improve pERK expression compared with 15 min (F=474.0, P＜0.05). The expression of HO-1 and Nrf2 proteins in the PSF+/U0126－ group was significantly more than those in the PSF－/U0126－ group, the expression of HO-1 and Nrf2 proteins in the PSF+/U0126+ group was significantly lower than that in the PSF+/U0126－ group, and the differences were statistically significant (F=30.2, 489.4; P＜0.05).ConclusionOver expression of PSF can promote the HO-1 expression by activating ERK pathway and promoting the Nrf2 to the nucleus, thus protect hRMECs against AGEs-induced oxidative damage.
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.