Wang Jie, Zhang Jingping, Han Shuang, Wang Youquan, Chen Liangbiao
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai, China.
International Research Center for Marine Biosciences, Ministry of Science and Technology, Shanghai Ocean University, Shanghai, China.
J Fish Biol. 2025 Apr;106(4):1134-1147. doi: 10.1111/jfb.16021. Epub 2024 Dec 10.
Takifugu obscurus (pufferfish) is an important species in aquaculture and has become widely popular in China and Japan. However, the development of the pufferfish aquaculture industry has been significantly impacted by severe diseases. Fish cell lines, as a model for in vitro studies, have the advantages of low cost, easy culture, and low genetic variation rate. A novel cell line from the fin of T. obscurus was established in this study and named TOF. TOF grew optimally with L-15 medium at 28°C and showed a multipolar fibroblast-like morphology. Mitochondrial cytochrome oxidase subunit 1 (COI) gene identification and karyotype detection confirmed that TOF cells originated from T. obscurus and the chromosome number was 44 (2n = 44). Transfection experiments showed that TOF cells were able to transfect and express exogenous genes by lipofection and electroporation. The studies of salinity (NaCl) and alkalinity (NaHCO) on TOF proliferation showed that the highest multiplication rate of TOF was obtained at 6‰ NaCl mass and 4 g/L alkalinity, respectively. Furthermore, the expressions of several genes associated with the immune response were significantly upregulated after stimulating TOF cells with lipopolysaccharide (LPS) and poly (I:C), including irf7, il10, myd88, and nod1. Additionally, the Aeromonas hydrophila infection assay with TOF cells showed that TOF cells were sensitive to bacteria, and pufferfish could promote antimicrobial activity through NLR/NF-κB pathway to regulate the production of cytokines. This study suggested that TOF cell line could be an advantageous in vitro model for the investigation of fish immunology and virology, and could assist us to better comprehend the T. obscurus immune response to bacterial invasion.
暗纹东方鲀（河豚）是水产养殖中的重要品种，在中国和日本广受欢迎。然而，河豚养殖业的发展受到严重疾病的显著影响。鱼类细胞系作为体外研究的模型，具有成本低、易于培养和遗传变异率低的优点。本研究建立了一种来自暗纹东方鲀鳍的新型细胞系，并命名为TOF。TOF在28°C下于L-15培养基中生长最佳，呈现多极成纤维细胞样形态。线粒体细胞色素氧化酶亚基1（COI）基因鉴定和核型检测证实TOF细胞源自暗纹东方鲀，染色体数为44（2n = 44）。转染实验表明，TOF细胞能够通过脂质体转染和电穿孔转染并表达外源基因。对TOF增殖的盐度（NaCl）和碱度（NaHCO）研究表明，TOF在NaCl质量分数为6‰和碱度为4 g/L时分别获得最高增殖率。此外，用脂多糖（LPS）和聚（I:C）刺激TOF细胞后，几种与免疫反应相关的基因表达显著上调，包括irf7、il10、myd88和nod1。此外，用嗜水气单胞菌感染TOF细胞的实验表明，TOF细胞对细菌敏感，河豚可通过NLR/NF-κB途径促进抗菌活性以调节细胞因子的产生。本研究表明，TOF细胞系可能是鱼类免疫学和病毒学研究的有利体外模型，并有助于我们更好地理解暗纹东方鲀对细菌入侵的免疫反应。
