过表达严重急性呼吸综合征冠状病毒2型刺突蛋白抑制人视网膜色素上皮细胞生长

Overexpression of SARS-CoV-2 spike protein mediates growth inhibition in human retinal pigment epithelial cells

目的观察过表达严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)刺突蛋白(S-蛋白)对人视网膜色素上皮(RPE)细胞生长的抑制作用。方法构建SARS-CoV-2S-蛋白基因片段表达质粒(p3xflag-S),并转染至ARPE-19、HEK293细胞内,应用DNA测序进行鉴定,并对表达的标签蛋白进行蛋白免疫印迹法(Western blot)检测。HEK293细胞分为细胞1、2、3、4,分别转染GFP11质粒和空载质粒、GFP1-10质粒和空载质粒、GFP11质粒和pCMV-HA-ACE2质粒、GFP1-10和p3xflag-S质粒。将细胞1与细胞2(对照组1)、细胞2与细胞3(对照组2)、细胞3与细胞4(观察组)、细胞1与细胞2、3、4(对照组3)混合共培养。明场显微镜和荧光显微镜观察细胞融合情况。将RPE细胞分为对照组、过表达S-蛋白组。流式细胞仪检测RPE细胞周期;计数试剂盒8(CCK-8)检测RPE细胞增生水平;Western blot检测RPE细胞中S-蛋白表达水平。组间比较行最小显著差法t检验。结果DNA序列测定结果显示,S-蛋白cDNA与标签蛋白融合;Western blot检测结果显示,与未转染p3xflag-S质粒的细胞比较,转染HEK293细胞中S-蛋白相关表达量升高。明场显微镜下可见大而多核的融合细胞团;荧光显微镜下可见融合细胞内有多个细胞核且呈明显绿色荧光。Western blot检测结果显示,与未转染p3xflag-S质粒的RPE细胞比较,转染p3xflag-S质粒的RPE细胞中S-蛋白相关表达量升高。CCK-8增生分析结果显示,与对照组比较,过表达S-蛋白组RPE细胞增生能力显著降低,差异有统计学意义(t-22.70、16.75、23.38,P<0.0001)。流式细胞仪检测结果显示,对照组、过表达S-蛋白组G1期细胞分别为41.1%、67.0%;与对照组比较,过表达S-蛋白组G1期细胞明显升高,差异有统计学意义(t=4.76,P=0.018)。过表达S-蛋白组细胞凋亡率较对照组明显增加,差异有统计学意义(t=4.91,P=0.008)。结论过表达SARS-CoV-2S-蛋白可抑制RPE细胞生长活性。

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,GFP1-10 plasmid 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 that S-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<0.0001).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 Gl 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.