The ultra.structural changes of photic injury to the retina of two patients caused by indirect ophthalmoscope were studied.The duration of exposure was 20 minutes. Under transmission electron microscope, dilation of the choroidal vessels,swelling and vacule formation at all retinal layers, disparsed pyknotic nuclei among the outer nuclear layer and swelling of the nerve fibers were found,The retina of the pregnant woman was injured more severely than that of the male patient. （Chin J Ocul Fundus Dis,1994,10:77-79）
Replacement of diseased retinal pigment epithelium (RPE) cells with healthy RPE cells by transplantation is one option to treat several retinal degenerative diseases including age-related macular degeneration, which are caused by RPE loss and dysfunction. A cellular scaffold as a carrier for transplanted cells, may hold immense promise for facilitating cell migration and promoting the integration of RPE cells into the host environment. Scaffolds can be prepared from a variety of natural and synthetic materials. Strategies, such as surface modification and structure adjustment, can improve the biomimetic properties of the scaffolds, optimize cell attachment and cellular function following transplantation and lay a foundation of clinical application in the future.
Objective To investigate the effect of blue light on mRNA expression of L-type calcium channel subtypes of human retinal pigment epithelial (RPE) cells in vitro. Methods The fourth-generation of human RPE cells were randomly divided into four groups including control group (no light group), light group, light + nifedipine group, and light + (-) BayK8644 group. The cells were exposed to blue light (2000plusmn;500) lux for 6 hours, and then cultured for another 24 hours. Reverse transcription polymerase chain reaction real time (RT-PCR) and fluorescence quantitative PCR technologies were used to analyze mRNA expression of L-type calcium channel subunit of cardiac subtype ( 1C or CaV1.2), neuroendocrine subtype ( 1D or CaV1.3) and retinal subtypes ( 1F or CaV1.4) in each group. Results The length of PCR product of 1C, 1D, 1F subunit and actin was 68, 157, 125 and 186 base pairs respectively. (1) 1C mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than that in control group, the difference was statistically significant (P＜0.05). 1C mRNA expression in light +nifedipine group and light + (-) BayK8644 group was higher than in light group (P＜0.05). 1C mRNA expression in light + (-) BayK8644 group was higher than that in light + nifedipine group (P＜0.05). (2) Comparing with control group, 1D mRNA expression was higher in light, light +nifedipine and light + (-) BayK8644 group, the difference was statistically significant (P＜0.05). Light + (-) BayK8644 group was higher than light group and light + nifedipine group (P＜0.05), light group and the light + nifedipine group was not statistically significant (P＞0.05). (3) 1F mRNA expression in light, light + nifedipine and light + (-) BayK8644 group was higher than those in control group, there was statistically significant (P＜0.05), light +nifedipine group and light + (-) BayK8644 group was higher than light group (P＜0.05), light + nifedipine group and the light + (-) BayK8644 group was not statistically significant (P＞0.05). Conclusions The human RPE cells mRNA expression of L-type calcium channel 1C, 1D and 1F subunit was increased after exposing to blue light. Application of the 1times;10-5 mmol/L (-) BayK8644 can increase mRNA expression of 1C, 1D and 1F subunit.
ObjectiveTo observe the clinical characterisitics of choroidal excavation in the macula. MethodsA total of 22 patients (22 eyes) with choroidal excavation diagnosed by spectral domain high definition optical coherence tomography (HD-OCT) were enrolled in this study. The patients included 12 males (54.50%) and 57 females (45.50%). The age was ranged from 21 to 82 years old, with an average of (41.44±13.17) years. All the patients were affected unilaterally, including 9 right eyes and 13 left eyes. The corrected vision, slit lamp microscope with preset lens, fundus photography, HD-OCT and fluorescence fundus angiography (FFA)were measured for all patients. The clinical characterisitics and concomitant diseases were observed. Seventeen eyes were followed for a period between 3 to 12 months. The lesions change were evaluated by HD-OCT. ResultsThere were 18 eyes (81.8%) with symptoms of micropsia and metamorphopsia, 4 eyes (18.2%) without symptoms. The corrected vision was ranged from 0.3 to 1.2, 12 eyes (54.54%) with moderate or high myopia. Fundus examination presents yellowish-white exudation in 12 eyes (54.54%), yellowish-white exudation accompanied with hemorrhage in 9 eyes (40.91%), grayish yellow reflex halo in 1 eye (4.55%). HD-OCT showed that the retinal pigment epithelium (RPE) layer was involved in the excavation, and the photoreceptor outer segment and pigment junction (OPR) layer was disappeared in all eyes. The external limiting membrane and the photoreceptor inner segment/outer segment junction layer were preserved in 13 eyes (59.09%) and disappeared in 9 eyes (40.91%). There were 10 eyes (18.18%) with a single lesion, 4 eyes (18.18%) with idiopathic choroidal neovascularization, 4 eyes (18.18%) with punctate inner choroidopathy, 1 eye (4.55%) with polypoidal choroidal vasculopathy, 1 eye (4.55%) with macular preretinal menbrance, 1 eye (4.55%) with central serous chorioretinopathy. FFA showed hypofluorescence in early phase, hyperfluorescence in late phase, without obvious leakage. There was no noticeable changes in size and morphological changes in the follow-up period. ConclusionsChoroidal excavation in the macula occurs mostly in middle-aged people with myopia. It can be associated with many fundus diseases. The excavation is located in RPE layer, and OPR layer disappeared. Choroidal excavation in the macula develops slowly.
Objective To study the inhibitive effect of adenovirus mediated CD gene and 5-FC on proliferative human retinal pigment epithelial (HRPE) cells, and to search for an effective method to take precautions against proliferative vitroretinopathy (PVR).Method Different concentrations of CD and 5-FC were added respectively to the cultured third-growth-generation HRPE cells.Transferance rate was detected by positive HRPE cells marked by X-gal and LacZ. The number of HRPE cells were counted and evaluated by methylthiazol-tetrazollium (MTT) method. Results The adenovirus mediated CD gene could be transfered into HRPE cells with a dose-dependent manner. Positive HRPE cells with CD gene could transform 5-FC to 5-Fu,which could inhibit the increase of HRPE cells effectively. No obvious bystander effect on the growth of HRPE cells was detected.Conclusions The adenovirus may introduce a foreign gene into cultured HRPE cells efficiently. It could be a good method to treat and prevent PVR by medication. (Chin J Ocul Fundus Dis，2003，19：168-171)
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.
Objective To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells. MethodsSARS-CoV-2 S-protein gene fragment expression plasmid (p3xflag-S) was constructed and transfected into human RPE, HEK293 cells. DNA sequencing was used for identification, and the expression of Flag-S was detected by Western blot. HEK293 cells were divided into the cells 1, 2, 3 and 4 and transfected with GFP11 plasmid and vector, GFP1-10plasmid and vector, transfected with GFP11 and pCMV-HA-ACE2 plasmid, GFP1-10 and p3xflag-S plasmid. Cell 1 was co-cultured with cell 2 (control group 1), cell 2 with cell 3 (control group 2), cell 3 with cell 4 (observation group), and cell 1 mixed with cells 2, 3 and 4 (control group 3). Bright-field microscopy and fluorescence microscopy were used to observe cell fusion. RPE cells were divided into control group and overexpression S-protein group. The cell cycle was detected by flow cytometry; the cell proliferation level was detected by Counting Kit 8 (CCK-8); and the S-protein expression level in RPE cells was detected by Western blot. The Student’s t-test was performed for comparison between groups. ResultsDNA sequence assay showed that S-protein cDNA was fused with flag-tagged protein. Western blot assay showed thatS-protein-related expression was elevated in transfected HEK293 cells compared with untransfected p3xflag-S cells. Large, multinucleated fused cell clusters were visible under bright-field microscopy; multiple nuclear with distinct green fluorescence were visible in the fused cells under fluorescence microscopy. Western blot assay showed elevated S-protein-related expression in transfected p3xflag-S plasmid RPE cells compared to untransfected p3xflag-S plasmid RPE cells. CCK-8 results showed that the proliferative capacity of RPE cells in the S-protein overexpression group was significantly reduced compared with the control group, with statistically significant differences (t=22.70, 16.75, 23.38; P<0.000 1). The results of flow cytometry showed that the G1 phase cells in the control and overexpression S-protein groups were 41.1% and 67.0%, respectively; compared with the control group, the G1 phase cells in the overexpression S-protein group were significantly higher, and the difference was statistically significant (t=4.76, P=0.018). The apoptosis rate was significantly increased in the S-protein overexpression group compared with the control group, and the difference was statistically significant (t=4.91, P=0.008). ConclusionOverexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.
Fluorescein angiography(FA)was performed in 31 pigmented rebbits.The angiograms were evaluated as prints and as negative film under a light microscope.The patterns of retinal pigment epithelial(RPE)cells were studied by scaning electron microscopy and fluorescein light one,compared with other rabbits belonging to the same species.In 58 eyes,we observed the hexagonal pattern of RPE cell.It showed central hypofluorescent area surrounded by hyperfluorescent rim,which was easily seen away from the medullary rays by three or more disc diameters and became larger in the periphery than that in the posterior pole.There were no finding in four lightly pigmented eyes. （Chin J Ocul Fundus Dis,1994,10:226-228）