Zhang Yuhang 1 , Xue Mengjiao 1 , Xie Xiaohang 2 , Hu Yanzhong 1 , Zhang Fengyan 1
  • 1. Ophthalmology Department of The First affiliated Hospital of Zhengzhou University, The Laboratory For Ophthalmology and Vision Science of Henan Eye Hospital, Zhengzhou 450052, China;
  • 2. College of Medicine, Zhengzhou University, Zhengzhou 4500052, China;
Zhang Fengyan, Email: zhangfengyanx@aliyun.com
SARS-CoV-2; Spike protein; Retinal pigment epithelium cells
10.3760/cma.j.cn511434-20211104-00630
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Objective  To observe the inhibition of SARS-CoV-2 spike protein (S-protein) on the proliferation of human retinal pigment epithelium (RPE) cells. Methods SARS-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. Results DNA 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). Conclusion Overexpression of the SARS-CoV-2 spike protein reduced the proliferation of human RPE cells.

Citation： Zhang Yuhang, Xue Mengjiao, Xie Xiaohang, Hu Yanzhong, Zhang Fengyan. Overexpression of SARS-CoV-2 spike protein mediates growth inhibition in human retinal pigment epithelial cells. Chinese Journal of Ocular Fundus Diseases, 2023, 39(3): 232-237. doi: 10.3760/cma.j.cn511434-20211104-00630 Copy

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