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.
ObjectiveTo observe the correlation between retinal capillary density and retinal thickness in the macula and spherical equivalent (SE) in school-age children. MethodsA cross-sectional study. From May to December 2022, 182 school-age children who visited the ophthalmology department of the First Affiliated Hospital of Zhengzhou University were included . There were 95 males and 87 females. The age ranged from 6 to 12 years, and the spherical equivalent (SE) was +0.50 to －6.00 D. They were divided into three groups based on the SE of the right eyes: 54 eyes in emmetropia group (+0.50≤SE＜－0.50 D), 71 eyes in low myopia group (－0.50≤SE＜－3.00 D), and 57 eyes in moderate myopia group (－3.00≤SE≤－6.00 D). The macular area of 6 mm×6 mm was scanned using swept-source optical coherence tomography angiography and was divided into three concentric rings centered on the fovea, including the macular central fovea (0-1 mm diameter), inner ring (1-3 mm diameter) and outer ring (3-6 mm diameter). The retinal thickness and blood flow density of superficial vascular plexus (SVP) and deep vascular plexus (DVP) in different zones within 6 mm of the macular area were measured. The relationships between SE and SVP, DVP and retinal thickness in each ring region were investigated by univariate and multivariate linear regression analyses, smooth curve fitting, and threshold effects. ResultsThere were significant differences in the SVP (F=6.64, 26.06, 22.69) and DVP (F=7.97, 25.01, 5.09) of macular central fovea, inner ring and outer ring among the emmetropia, low myopia and moderate myopia groups (P＜0.05). Univariate linear regression analysis showed that the SVP (β=－0.56, －1.17, －0.79) and DVP (β=－1.03, －0.93, －0.45) of the three regions were positively correlated with SE (P＜0.05). After smooth curve fitting, threshold effect analysis and multivariate linear regression analysis, the SVP and DVP in the macular central fovea were linearly positively correlated with SE (β=－0.91, －1.40; P＜0.05), and SVP and DVP in the inner ring and outer ring showed an inverted U-shaped curve relationship with SE with the inflection (＜3.00 D). When the SE was less than ＜3.00 D, the SVP and DVP in the inner ring and outer ring were positively correlated with SE (P＜0.05). When the SE was higher than －3.00 D, except for the DVP in the inner ring region, the other parameters were negatively correlated with SE (P＜0.05). There were significant differences in retinal thickness of the inner ring and outer ring (F=5.47, 16.36; P＜0.05), and no significant difference in the macular central fovea among the emmetropia, low and moderate myopia groups (F=2.16, P＞0.05). By using univariate and multivariate linear regression analyses, the retinal thickness in the inner ring and outer ring were negatively correlated with SE (β =1.99, 3.05; P＜0.05). However, no correlation was found between retinal thickness and SE in the macular central fovea (β=－1.65, P＞0.05). ConclusionsIn school-age children with SE between +0.50 D and －6.00 D, the retinal capillaries density of the macular central fovea gradually increase, and increase first and then decrease in the inner ring and outer ring with increasing SE. The retinal thickness of inner ring and outer ring gradually decrease and not change significantly in the macular central fovea.