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
1. Institute of Medical Biology, Chinese Academy of Medical Sciences/ Peking Union Medical College,Kunming 650118, China; 2. Jinhua Central Hospital, Jinhua 321099, China; 3. Kunming Medical University, Kunming 650500, China
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 HSV1(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 × 104 CCID50 / 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 type Ⅱ, 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 type Ⅱ, 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.
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.
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