SARS⁃2⁃S诱导形成合胞体促进A549和B16细胞体外增殖和迁移  

Syncytia formation induced by SARS⁃2⁃S promotes the proliferation and migration of A549 and B16 cells in vitro

作　　者：周丽[1,2] 钟辉 万禄明 周鹏宇 刘慕仪 魏从文 周传意 ZHOU Li;ZHONG Hui;WAN Luming;ZHOU Pengyu;LIU Muyi;WEI Congwen*;ZHOU Chuanyi(Yueyang People's Hospital Affiliated to Hunan Normal University,Yueyang,Hunan 414000,China;Laboratory of Advanced Biotechnology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)

机构地区：[1]湖南师范大学附属岳阳市人民医院,湖南岳阳414000 [2]军事科学院军事医学研究院前沿生物技术实验室,北京100071

出　　处：《军事医学》2024年第12期881-888,共8页Military Medical Sciences

基　　金：湖南省自然科学基金项目(2023JJ50297)。

摘　　要：目的研究严重急性呼吸综合征冠状病毒2(SARS-CoV-2)的刺突蛋白(SARS-2-S)诱导形成合胞体对人类肺腺癌细胞A549和小鼠黑色素瘤细胞B16体外增殖和迁移的影响。方法构建表达人血管紧张素转化酶2(hACE2)和SARS-2-S的质粒,分别和慢病毒包装质粒转染HEK-293FT细胞,收集含慢病毒的上清感染A549细胞,嘌呤霉素筛选后得到稳定转染hACE2细胞系(A549-A)和SARS-2-S细胞系(A549-S),Western印迹实验验证A549-A和A549-S蛋白表达情况,将A549-A和A549-S细胞共培养后用荧光显微镜观察合胞体。使用条件培养基(合胞体上清和非合胞体上清)体外培养A549、鸡卵清蛋白(OVA)基因修饰的B16细胞(B16-OVA)、B16-F10后,通过CCK-8实验、集落形成实验检测肿瘤细胞的体外增殖能力,通过划痕实验检测肿瘤细胞的体外迁移能力。结果成功构建分别稳定转染hACE2和SARS-2-S的A549细胞系;将A549-A和A549-S细胞共培养后建立SARS-2-S诱导形成合胞体体系。合胞体上清和非合胞体上清均能显著促进A549、B16-OVA、B16-F10细胞的体外增殖、迁移能力,但合胞体上清比非合胞体上清的作用更加明显。结论SARS-2-S诱导形成合胞体促进A549和B16细胞的体外增殖和迁移能力。Objective To investigate the effects of syncytial formation induced by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)spike protein(SARS-2-S)on the proliferation and migration of human lung adenocarcinoma A549 cells and mouse melanoma B16 cells in vitro.Methods Plasmids expressing human angiotensin-converting enzyme 2(hACE2)and SARS-2-S were constructed and respectively co-transfected with lentiviral packaging plasmids into HEK-293FT cells before the lentiviral supernatant was collected and infected with A549 cells which were screened by puromycin to obtain the A549 cells respectively that were stably transfected with hACE2(A549-A)and SARS-2-S(A549-S).The protein expression of A549-A and A549-S cells was verified by Western blotting.A549-A and A549-S cells were co-cultured before their syncytia were observed by fluorescence microscopy.Conditioned media(syncytial supernatant and non-syncytial supernatant)was collected to culture A549 cells,ovalbumin(OVA)-gene-modified B16 cells(B16-OVA),and B16-F10 cells in vitro.The CCK-8 assay and colony formation assay were used to assess the proliferation capacity of tumor cells,while the wound healing assay was employed to evaluate the migration capacity of tumor cells.Results Stable A549 cell lines expressing hACE2 and SARS-2-S were constructed.The SARS-2-S-induced syncytial formation system was established after co-culture of A549-A and A549-S cells.Both syncytial and non-syncytial supernatants significantly promoted the proliferation and migration of A549,B16-OVA,and B16-F10 cells in vitro,especially the syncytial supernatant.Conclusion SARS-2-S-induced syncytial formation promotes the proliferation and migration properties of A549 and B16 cells in vitro.

关 键 词：严重急性呼吸综合征冠状病毒2 刺突蛋白 合胞体 细胞增殖 细胞迁移 

分 类 号：R373[医药卫生—病原生物学]