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

作　　者：丁相荣 陈柳[1,2] 霍姝汭 杞梦迪 刘欣 王文广 李娜[1] 代解杰[1] 陆彩霞[1] DING Xiangrong;CHEN Liu;HUO Shurui;QI Mengdi;LIU Xin;WANG Wenguang;LI Na;DAI Jiejie;LU Caixia(Institute of Medical Biology,Chinese Academy of Medical Sciences/Peking Union Medical College,Kunming 650118,China;Jinhua Central Hospital,Jinhua 321099,China;Kunming Medical University,Kunming 650500,China)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所,昆明650118 [2]金华市中心医院,浙江金华321099 [3]昆明医科大学,昆明650500

出　　处：《中国实验动物学报》2024年第5期610-619,共10页Acta Laboratorium Animalis Scientia Sinica

基　　金：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)。

摘　　要：目的建立树鼩永生化角膜基质细胞(corneal stromal cells,CSCs)系,并探讨其在病毒感染中的应用。方法利用组织块贴壁培养法分离培养树鼩原代CSCs,将携带SV40T基因的慢病毒转染细胞后,再挑取单克隆进行传代培养,传至50代以上进行形态学观察并与40代细胞形态进行比较,波形蛋白(vimentin)和SV40T基因免疫荧光鉴定、核型鉴定、细胞增殖曲线测定。用单纯疱疹病毒1型(herpes simplex virus-1,HSV-1)(McKrae株)、寨卡病毒(Zika virus,ZIKV)(GZ01株)、登革病毒Ⅱ型以及甲型流感病毒H1N1(PR8株)在该细胞上进行病毒感染实验。结果传至50代以上的树鼩永生化CSCs呈梭形,细胞形态结构与40代相比仍较好。vimentin和SV40T基因免疫荧光表达阳性。增殖曲线结果显示:细胞生长旺盛,第4~5天时处于对数生长期。原代细胞核型的染色体数稳定为62条,而永生化细胞第21、56代突变为64条且保持稳定。病毒感染实验显示:树鼩永生化CSCs对HSV-1(McKrae株)、ZIKV(GZ01株)、登革病毒Ⅱ型以及H1N1(PR8株)病毒敏感,产生较高感染滴度,分别为1.32×105、5.62×106、2.69×107、7.76×104CCID50/mL。结论成功建立了树鼩永生化CSCs细胞系,提示该细胞系可用于单纯疱疹病毒、寨卡病毒、登革病毒和甲型流感病毒感染眼角膜疾病的作用机制及抗病毒药物的研究。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、7.76×104 CCID50/mL,respectively.Conclusions Theimmortalized 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 

分 类 号：Q95-33[生物学—动物学]