严重急性呼吸综合征冠状病毒2型感染对小鼠视网膜光感受器细胞形态、增殖、凋亡及免疫功能的影响  

Effects of SARS-CoV-2 infection on the morphology,proliferation,apoptosis and immune function of retinalphotoreceptorcellsinmice

作　　者：席懿璇 窦国睿 周子义 常天芳 储昭节 Xi Yixuan;Dou Guorui;Zhou Ziyi;Chang Tianfang;Chu Zhaojie(College of Life Sciences,Northwestern University,Xi'an 710000,China;Department of Ophthalmology,First Affiliated Hospital of Northwest University,Xian First Hospital,Xi'an 710002,China;Department of Ophthalmology,Eye Institute of Chinese PLA,Fourth Military Medical University,Xi'an 710032,China;Shaanxi Eye Research Institute,Xi'an 710032,China)

机构地区：[1]西北大学生命科学学院,西安710000 [2]西北大学附属第一医院西安市第一医院,西安710002 [3]空军军医大学附属第一医院眼科,西安710032 [4]陕西省眼科研究所,西安710021

出　　处：《中华眼底病杂志》2024年第10期772-780,共9页Chinese Journal of Ocular Fundus Diseases

基　　金：国家自然科学基金(82371071、81970814、82000905);西安市科技计划项目(22YXYJ0053)。

摘　　要：目的观察严重急性呼吸综合征冠状病毒2型(SARS-CoV-2)感染对小鼠视网膜光感受器细胞(661w细胞)形态、增殖、凋亡、细胞周期及免疫应答功能的影响。方法细胞实验。将体外培养的对数生长期661w细胞, 利用血管紧张素转化酶2(ACE2)过表达慢病毒转染细胞, 构建可感染SARS-CoV-2假病毒(以下简称为"假病毒")的ACE2过表达661w细胞。将661w细胞分为三组, 分别为正常组(未经任何处理)、siACE2组(过表达ACE2且未感染假病毒)及感染组(过表达ACE2并感染假病毒), 其中感染组分为5 TU/ml假病毒组、15 TU/ml假病毒组、30 TU/ml假病毒组、50 TU/ml假病毒组, 分别感染12、24、48、72 h。荧光显微镜观察ACE2转染效率;蛋白质免疫印迹法(Western blot)检测细胞中ACE2相对表达水平;光学显微镜观察正常组与感染组细胞形态;细胞计数试剂盒-8(CCK8)法检测细胞增殖;流式细胞仪检测细胞周期;Western blot、实时定量聚合酶链反应(qPCR)检测siACE2组、感染组(30 TU/ml假病毒组)细胞中白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白和mRNA相对表达量;qPCR检测siACE2组、感染组(30 TU/ml假病毒组)细胞中核因子(NF)-κB-1、NF-κB2以及NF- κB增强子(P65)、前体蛋白(P100)mRNA相对表达量。多组间比较采用单因素方差分析;两组间比较采用t检验。结果与siACE2组比较, 感染组细胞均出现不同程度皱缩, 随假病毒诱导浓度、时间增加, 细胞皱缩加重, 细胞数量减少。与正常组比较, 感染组细胞随假病毒诱导时间延长, 细胞存活率逐渐降低, 差异无统计学意义(F=0.840、0.412、1.498、1.138, P>0.05), 与siACE2组比较, 30、50 TU/ml假病毒组诱导细胞后凋亡指数显著升高, 差异有统计学意义(F=2.523、6.716、3.477、3.421, P<0.05)。假病毒诱导72 h时, 与siACE2组比较, 30 TU/ml假病毒组G1期细胞显著增加, 差异有统计学意义(t=3.8Objective 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(661w 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 pseudovirus group,and the cells were infected with the pseudovirus 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)-kBI and NF-kB2,as well as NF-kB enhancer(P65)and precursor protein(P100)in cells of the siACE2 group and the infected group(30 TU/

关 键 词：急性呼吸综合征冠状病毒2型 光感受器细胞 细胞凋亡 细胞增殖 

分 类 号：R511.9[医药卫生—内科学]