Effects of SARS-CoV-2 infection on the morphology,proliferation,apoptosis and immune function of retinal photoreceptor cells in mice
Objective To observe the effects of SARS-CoV-2 infection on the morphology,proliferation,apoptosis,cell cycle,and immune response function of mouse retinal photoreceptor cells(66 1w cells).Methods A cell experiment.Logarithmic growth phase 661w cells were cultured in vitro and transfected with angiotensin-converting enzyme 2(ACE2)overexpressing lentivirus to construct ACE2 overexpressing 661w cells that could be infected with SARS-CoV-2 pseudovirus(hereafter referred to as'pseudovirus').The 661w cells were divided into three groups:the normal group(untreated),the siACE2 group(overexpressing ACE2 and not infected with the pseudovirus)and the infected group(overexpressing ACE2 and infected with the pseudovirus),in which the infected group was 5 TU/ml pseudovirus group,15 TU/ml pseudovirus group,30 TU/ml pseudovirus group and 50 TU/ml pseudo virus group,and the cells were infected with the pseudo virus for 12,24,48 and 72 h,respectively.The infected group was infected with 5 TU/ml pseudovirus group,15 TU/ml pseudovirus group,30 TU/ml pseudovirus group and 50 TU/ml pseudovirus group,respectively,for 12,24,48 and 72 h.Fluorescence microscopy was used to observe the transfection efficiency of ACE2;protein immunoblotting(Western blot)was used to detect the relative expression level of ACE2 in the cells;light microscope was used to observe the morphology of the cells in the normal and the infected groups;cell proliferation was detected by Cell Counting Kit-8(CCK8)assay;flow cytometry was used to detect the cell cycle;Western blot and real-time quantitative polymerase chain reaction(qPCR)were used to detect the relative expression of interleukin-6(IL-6),tumour necrosis factor-α(TNF-α),B lymphocytoma-2(Bcl-2),Bcl-2-associated X-protein(Bax)proteins and mRNA in the cells of siACE2 group,infected group(30 TU/ml pseudovirus group);qPCR was used to detect the relative expression of nuclear factor(NF)-κBI and NF-κB2,as well as NF-κB enhancer(P65)and precursor protein(P1 00)in cells of the siACE2 group and the infected group(30 TU/ml pseudovirus group).One-way ANOVA was used for comparison between multiple groups;t-test was used for comparison between two groups.Results Compared with the siACE2 group,the cells in the infected group showed different degrees of crumpling,and with the increase of the concentration and time of pseudovirus induction,the crumpling of the cells worsened,and the number of cells decreased.Compared with the normal group,the cells in the infected group showed a gradual decrease in cell viability with the prolongation of pseudovirus induction time,and the difference was no statistically significant(F=0.840,0.412,1.498,1.138;P＞0.05),and the apoptotic index of the cells induced in the 30 and 50 TU/ml pseudovirus group was significantly elevated,and the difference was statistically significant(F=2.523,6.716,3.477,3.421;P＜0.05).At 72 h of pseudovirus induction,compared with the siACE2 group,the G1 phase cells in the 30 TU/ml pseudovirus group were significantly increased,and the difference was statistically significant(t=3.812,P＜0.05);the relative expression of IL-6,TNF-α,Bax protein and mRNA in the cells was up-regulated(t=7.601,6.039,3.088,5.193,6.427,7.667;P＜0.05),the relative expression of Bcl-2 protein and mRNA was down-regulated(t=3.614,6.777;P＜0.05),and the relative expression of NF-κB1,NF-κB2,P65,and P100 mRNA was significantly up-regulated with statistically significant differences(t=3.550,3.074,3.307,4.218;P＜0.05).Conclusion SARS-CoV-2 infection may inhibit photoreceptor cell proliferation,promote apoptosis and cycle blockade by activating the NF-κB signalling pathway.
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