背景:原代绵羊胎儿成纤维细胞(SFFC)已成为研究口蹄疫病毒(ORFV)的分子和致病机制的宝贵资源。然而,由于与它们的隔离和培养相关的高昂费用,它们的利用受到相当大的限制,它们缩短的寿命,和费力的程序。
结果:在我们的调查中,通过引入含有猿猴病毒40(SV40)的大T抗原的慢病毒重组质粒,获得了原代SFFC并使其永生化。纤维连接蛋白和波形蛋白的表达,SV40大T抗原的活性,细胞增殖试验,和程序性细胞死亡的分析表明,永生化大T抗原SFFCs(TSFFCs)保持着与初级SFFCs相同的生理特征和生物学功能。此外,TSFFC对细胞凋亡表现出强大的抵抗力,延长寿命,与原发性SFFC相比,增殖活性增强。值得注意的是,在裸鼠中,原代SFFC未经历体外转化或表现出任何恶性肿瘤的迹象.此外,永生化的TSFFCs显示ORFV活疫苗易感性.
结论:永生化的TSFFC提供了有价值的体外模型,用于使用各种技术探索ORFV的特征。这表明它们在涉及病毒分离的未来研究中具有安全利用的潜力,疫苗开发,和药物筛选。
BACKGROUND: Primary sheep fetal fibroblasts (SFFCs) have emerged as a valuable resource for investigating the molecular and pathogenic mechanisms of orf viruses (ORFV). However, their utilization is considerably restricted due to the exorbitant expenses associated with their isolation and culture, their abbreviated lifespan, and the laborious procedure.
RESULTS: In our investigation, the primary SFFCs were obtained and immortalized by introducing a lentiviral recombinant plasmid containing the large T antigen from simian virus 40 (SV40). The expression of fibronectin and vimentin proteins, activity of SV40 large T antigen, cell proliferation assays, and analysis of programmed cell death revealed that the immortalized large T antigen SFFCs (TSFFCs) maintained the same physiological characteristics and biological functions as the primary SFFCs. Moreover, TSFFCs demonstrated robust resistance to apoptosis, extended lifespan, and enhanced proliferative activity compared to primary SFFCs. Notably, the primary SFFCs did not undergo in vitro transformation or exhibit any indications of malignancy in nude mice. Furthermore, the immortalized TSFFCs displayed live ORFV vaccine susceptibility.
CONCLUSIONS: Immortalized TSFFCs present valuable in vitro models for exploring the characteristics of ORFV using various techniques. This indicates their potential for secure utilization in future studies involving virus isolation, vaccine development, and drug screening.