中国实验动物学报2024，Vol.32Issue(5)：610-619,10.DOI:10.3969/j.issn.1005-4847.2024.05.008
树鼩角膜基质永生化细胞系的构建及其在病毒感染性方面的研究
Construction of immortalized tree shrew corneal stromal cell line and investigation of viral infectivity
摘要
Abstract
Objective To establish an immortalized tree shrew corneal stromal cells(CSCs)line and to study its response to virus infection.Methods Primary tree shrew CSCs were isolated and cultured by the tissue block adhesion method.CSCs were then transfected with a lentivirus carrying the SV40T gene and monoclonal cells were selected for passage culture.The characteristics of the CSCs were investigated by morphological observation and compared with 40 generations until the 50 generations or more,immunofluorescence identification of vimentin and SV40T genes,karyotype examination,and cell proliferation curve.The CSCs were infected with herpes simplex virus-1(HSV-1)(McKrae strain),Zika virus(ZIKV,GZ01 strain),Dengue virus typeⅡ,and H1N1(PR8).Results The immortalized tree shrew CSCs after＞50 passages appeared spindle-shaped with good cell morphology and structure compared with 40 generations.Positive immunofluorescence expression of vimentin and SV40T genes.The cell growth curve showed that the cells were in logarithmic-phase growth on days 4～5 and grew vigorously.The number of chromosomes in the primary cells was stable at 62,while immortalized CSCs had 64 chromosomes at P21 and P56.The virus titer results showed that the immortalized tree shrew CSCs were sensitive to HSV-1(McKrae strain),ZIKV(GZ01 strain),Dengue virus typeⅡ,and H1N1(PR8),with virus titers of 1.32×105,5.62×106,2.69×107,and 7.76×104 CCID50/mL,respectively.Conclusions The immortalized tree shrew CSCs were established successfully,suggesting that this cell line is suitable for studies of the mechanisms of HSV,ZIKV,Dengue virus,and influenza A virus infection in relation to corneal diseases and antiviral drugs.关键词
树鼩/角膜基质细胞/永生化/病毒扩增Key words
tree shrew/corneal stromal cell/immortalized cell line/virus amplification分类
生物科学引用本文复制引用
丁相荣,陈柳,霍姝汭,杞梦迪,刘欣,王文广,李娜,代解杰,陆彩霞..树鼩角膜基质永生化细胞系的构建及其在病毒感染性方面的研究[J].中国实验动物学报,2024,32(5):610-619,10.基金项目
国家重点研发计划项目(2021YFF0702404),云南省基础研究计划项目(202101AT070291),云南省眼科疾病防治研究重点实验室(2017DG008),昆明市科技创新团队(2019-1-R-24483).Funded by the National Key R&D Program of China(2021YFF0702404),Yunnan Fundamental Research Projects(202101AT070291),Yunnan Key Laboratory of Eye Disease Prevention and Treatment(2017DG008),Kunming Science and Technology Innovation Team(2019-1-R-24483). （2021YFF0702404）
