Establishment and characterization of a liver cell line from black porgy, Acanthopagrus schlegelii
Establishment and characterization of a liver cell line from black porgy, Acanthopagrus schlegelii
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DOI：
10.1016/j.aqrep.2022.101213
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发表时间：
2022-08
影响因子：
3.7
通讯作者：
Hongbo Pan;Huiquan Chen;Leshi Chen;Wanwan Zhang;P. Lan;Peng Jia;Shizu Li;M. Yi;K. Jia
中科院分区：
文献类型：
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作者：
Hongbo Pan;Huiquan Chen;Leshi Chen;Wanwan Zhang;P. Lan;Peng Jia;Shizu Li;M. Yi;K. Jia
在这项研究中，肝细胞系（ASL）来自黑鲷（许氏鲷）的建立和特点。在初始原代培养后，将细胞系在28 °C下在含有15%胎牛血清（FBS）的Dulbecco改良Eagle培养基（DMEM）中培养超过40代。免疫组化结果表明，ASL细胞在培养初期至少含有两种细胞型，但在第6 ~ 20代，上皮样细胞占优势，并取代了另一种细胞型。ASL细胞在20-28 °C的温度范围内在含有15- 20%FBS的DMEM中生长。细胞遗传学分析表明，ASL细胞的众数为48。细胞色素c氧化酶亚基I基因的部分扩增表明ASL起源于黑鲷。用pEGFP-N3质粒转染ASL细胞后，转染效率约为15%。红点石斑鱼神经坏死病毒（RGNNV）感染后，在ASL细胞中观察到细胞病变效应，并使用定量逆转录聚合酶链反应，病毒滴度测定，蛋白质印迹和透射电子显微镜测定证实在RGNNV感染的ASL细胞中的病毒复制。此外，在RGNNV感染的ASL细胞中，几种抗病毒基因被不同程度地诱导，表明RGNNV感染后ASL细胞中的免疫应答被激活。总之，新建立的ASL细胞系是研究NNV-黑鲷相互作用的理想体外工具。
In this study, a liver cell line (ASL) derived from black porgy (Acanthopagrus schlegelii) was established and characterised. After the initial primary culture, the cell line has been cultured for over 40 passages at 28 °C in Dulbecco's modified Eagle's medium (DMEM) with 15 % foetal bovine serum (FBS). Immunostaining results indicated that ASL cells contained at least two cytotypes in the initial culture stage; however, epithelioid cells dominated and replaced the other type during passage 6–20. ASL cells grow in temperature range 20–28 °C in DMEM containing 15–20 % FBS. Cytogenetic analysis showed that the modal chromosome number in ASL cells was 48. Partial amplification of the cytochrome c oxidase subunit I gene indicates that ASL originates from black porgy. ASL cells showed a transfection efficiency of approximately 15 % after transfection with thepEGFP-N3plasmid. Cytopathic effects were observed in ASL cells after red-spotted grouper nervous necrosis virus (RGNNV) infection, and viral replication in RGNNV-infected ASL cells was confirmed using quantitative reverse transcription polymerase chain reaction, virus titre assays, western blot, and transmission electron microscopy assays. Furthermore, several antiviral genes were induced to varying degrees in RGNNV-infected ASL cells, indicating that the immune response was activated in ASL cells post RGNNV infection. In conclusion, the newly established ASL cell line is an idealin vitrotool for studying NNV-black porgy interactions.