日本鳗鲡心脏细胞系的建立及其在鳗鲡免疫应答研究中的应用

ESTABLISHMENT OF A JAPANESE EEL HEART CELL LINE AND ITS APPLICATION IN EEL IMMUNE RESPONSE RESEARCH

  • 摘要: 为建立稳定传代的鳗鲡细胞系用于鳗鲡免疫抗病机制的研究, 研究利用组织块法建立了日本鳗鲡(Anguilla japonica)心脏细胞系(Anguilla japonica Heart cells, AJH)。AJH细胞在28℃、含20%胎牛血清(FBS)的DMEM/F-12培养基中培养, 传代时间间隔3—4d, 在第5代后更换为15% FBS传代培养。核糖体RNA基因分析显示细胞来源于日本鳗鲡。核型分析显示AJH细胞具有38条染色体, 其中10条为中部着丝点染色体, 10条为近中部着丝点染色体, 18条为端着丝点染色体。病毒感染实验结果显示, 鳗鲡疱疹病毒(Anguillad herpesvirus, AngHV)可在AJH细胞中增殖。利用多种试剂转染EGFP-AjLC3至AJH细胞, 并通过流式细胞仪检测转染效率, 结果显示AJH最高转染效率为(37.17±0.12)%。将AjIRF1真核表达质粒和AjIFNaAjIFNc1启动子共转染至AJH细胞, 利用双荧光素酶报告分析了过表达AjIRF1对AjIFN启动子荧光素酶活性的影响。结果显示AjIFNaAjIFNc1启动子活性被显著上调, 表明AJH细胞可用于体外的基因功能研究。经LPS刺激后, AJH细胞中AjTNF-αAjIL-Iβ的表达水平明显上调; 转染Poly I:C后, AJH细胞中AjRIG-IaAjRIG-Ib的表达水平明显上调, 表明LPS和Poly I:C可诱导AJH细胞产生免疫应答。综上, 研究所建立的日本鳗鲡心脏细胞系可为深入研究鳗鲡抗病毒、抗细菌免疫应答机制提供细胞实验模型。

     

    Abstract: In order to establish a stable eel cell line conducive for studying eel immune response, we utilized the tissue block method to isolate and cultivate a heart cell line from Japanese eel (Anguilla japonica). Designated as AJH (Anguilla japonica Heart cells, AJH), these cells were cultured in DMEM/F-12 medium containing 20% fetal bovine serum (FBS) at 28℃, with passages conducted every 3 to 4 days. Subsequent to the 5th generation, cells were maintained in DMEM/F-12 medium with 15% FBS for ongoing passage culture. Ribosomal RNA analysis confirmed the Japanese eel origin of the cells. Karyotype analysis revealed that AJH cells possessed 38 chromosomes, comprising 10 metacentric, 10 submetacentric, and 18 acrocentric chromosomes. Viral infection experiments showed efficient propagation of Anguilla herpesvirus 1 (AngHV-1) within AJH cells. EGFP-AjLC3 transfection into AJH cells was carried out using a variety of reagents, and transfection efficiency was detected by flow cytometry, with the highest observed transfection efficiency in AJH cells reaching (37.17±0.12)%. In addition, co-transfection of AjIRF1 with AjIFNa or AjIFNc1 promoters into AJH cells resulted in up-regulation of AjIFNa or AjIFNc1 promoter activities, indicating the potential utility of AJH cells in gene function studies. After LPS stimulation, expression levels of AjTNF-α and AjIL-Iβ in AJH cells were significantly up-regulated. Similarly, after Poly I:C transfection, expression levels of AjRIG-Ia and AjRIG-Ib were significantly up-regulated, suggesting the inducible nature of immune responses in AJH cells by LPS and Poly I:C. Overall, the establishment of the Japanese eel heart cell line in this study presents a valuable cellular model for elucidating the mechanisms underlying antiviral and antibacterial immune responses in eels.

     

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