|
|
|
| Establishment and Characterization of a Liver Cell Line from Spotted Knifejaw (Oplegnathus punctatus) |
| ZHANG Hong-Xiang1,2, LI Kun-Ming1,2, WANG Na1, ZHAI Jie-Ming3, CHEN Song-Lin1* |
1 Yellow Sea Fisheries Research Institute, Chinese Academy of Fisheries Sciences, Function Laboratory for Marine Fisheries Science and Food Production Processes/Qingdao National Laboratory for Ocean Science and Technology, Qingdao 266071, China;
2 College of Fisheries and Life Sciences, Shanghai Ocean University, Shanghai 201306, China;
3 Shandong Laizhou Mingbo Aquatic Products Co., Ltd., Yantai 261400, China |
|
|
|
|
Abstract As powerful tool, fish cell lines have been widely used in virology, environmental toxicology, cell biology, genomics and genetics. Spotted knifejaw (Oplegnathus punctatus) is an excellent marine fish species. In this study, the tissue block method was used to establish the O. punctatus liver cell line (OPL) from spotted knifejaw liver. The culture medium used for the cell line was L-15 which was supplemented with mycillin, fetal bovine serum (FBS), antibiotics, basic fibroblast growth factor (bFGF) , and 2-mercaptoethanol (2-ME) . The cultured OPL cells, fibroblastic in morphology, had been subcultured to more than 30 passages in a good proliferating state. By comparing the effects of different medium of L-15 (Leibovitz-15), DMEM/F12 (Dulbecco's modified Eagle medium/Ham's F-12), DMEM and MEM on cell growth, the result showed that the optimum medium was L-15. The effects of temperature, FBS and bFGF on the growth of OPL cells were examined. The cells grew well in the temperature of 18~30 ℃, and the optimum growth temperature was 24 ℃. The results showed that the significant growth rate of OPL cells were accompanied with the increase of serum concentration range of 0%~20%. In addition, bFGF was benefit for growth of OPL cells. Chromosome analysis indicated that the passage 20 cells exhibited a normal diploid karyotype (2n=2sm+46t), but the ratio was only 68%. And the significant fluorescent signals were detected in OPL cells after transfection with pEGFP-N3 plasmid. The cytochrome oxidase subunitⅠ (COⅠ) gene was used to identified that OPL cell line originated from O. punctatus. Moreover, the establishment of the OPL cell line could provide basic experimental materials for studying the mechanisms of infective viruses and the function analysis of cells and genes.
|
|
Received: 25 September 2019
|
|
|
|
Corresponding Authors:
*chensl@ysfri.ac.cn
|
|
|
|
[1] 陈松林, 秦启伟. 2011. 鱼类细胞培养理论与技术[M]. 科学出版社, 北京. 3-16.
(Chen S L, Chen Q W.2013. Fish Cell Culture Theory and Technology[M]. Science Press, Beijing, 3-16.)
[2] 贾睿, 曹丽萍, 丁炜东, 等. 2012. 鱼类肝细胞分离、原代培养与应用研究综述[J]. 江西农业大学学报, 34(1): 147-157.
(Jia R, Cao L P, Ding W D, et al.2012. Isolation, primary culture and application of fish hepatocytes: An overview[J]. Acta Agriculturae Universitis Jiangxiensis, 34(1): 147-157. )
[3] 刘肖峰, 陈松林, 沙珍霞, 等. 2015. 云纹石斑鱼心脏细胞系的建立与鉴定[J]. 农业生物技术学报, 23(10): 1394-1400.
(Liu X F, Chen S L, Sha Z X.2015. Establishment, characterization of a new cell line from heart of kelp bass (Epinephelus moara)[J]. Journal of Agricultural Biotechnology, 23(10): 1394-1400.)
[4] 骆源, 张春晓, 王玲, 等. 2016. 斜带石斑鱼肝细胞分离及原代培养方法的建立[J]. 水产学报, 40(4): 558-565.
(Luo Y, Zhang C X, Wang L, et al.2016. Study on the isolation and primary culture of hepatocytes from liver of grouper (Epinephelus coioides), 40(4): 558-565.)
[5] 任国诚. 2007. 几种重要海水养殖鱼类细胞系的建立鉴定及应用[D]. 博士学位论文, 中国海洋大学, 导师: 陈松林, pp. 63-73.
(Ren G C.2007. The establishment, identification and application of cell lines from several important marine fish abstract[D]. Thesis for Ph.D., Ocean University of China, Suppervisor: Chen S L, pp. 63-73.)
[6] 任国诚, 陈松林, 沙珍霞. 2008. 半滑舌鳎肝脏细胞系的建立与鉴定[J]. 高技术通讯, 18(6): 657-660.
(Ren G C, Chen S L, Sha Z X.2008. Development and characterization of a liver cell line from half-smooth tongue-sole (Cynoglossus semilaevis)[J]. Chinese High Technology Letters, 18(6): 657-660.)
[7] 王梦珣, 朱鹤, 王玉珏, 等. 2018. 斑石鲷肌肉、脑细胞系的建立与生物学特性[J]. 中国海洋大学学报(自然科学版), 48(6): 67-74.
(Wang M X, Zhu H, Wang Y J, et al.2018. Establishment and characterization of two cell lines derived from spotted knifejaw Oplegnathus punctatus muscle and brain[J]. Periodical of ocean university of china (Natural science), 48(6): 67-74. )
[8] 薛蕊, 安皓, 刘清华,等. 2016. 斑石鲷(Oplegnathus punctatus)雌、雄鱼核型及Ag-NORs带型分析[J]. 海洋与湖沼, 47(3): 626-632.
(Xue R, An H, Liu Q H, et al.2016. Karyotype and Ag-NORs in male and female of Oplegnathus punctatus[J]. Oceanologia Et Limnologia Sinica, 47(3): 626-632.
[9] 张博, 陈松林. 2011. 近10年鱼类细胞培养研究进展及应用展望[J]. 海洋科学, 35(7): 113-121.
(Zhang B, Chen S L.2011. Decades of researching progresses of fish cell culture and its application prospects[J]. Marine Sciences, 35(7): 113-121.)
[10] Bradford C S, Sun L, Barnes D W.1994. Basic fibroblast growth factor stimulates proliferation and suppresses melanogenesis in cell cultures derived from early zebrafish embryos[J]. Molecular Marine Biology and Biotechnology , 3(2): 78-86.
[11] Block G D, Locker J, Bowen W C, et al.1996. Population expansion, clonal growth, and specific differentiation patterns in primary cultures of hepatocytes induced by HGF/SF, EGF and TGF alpha in a chemically defined (HGM) medium[J]. The Journal of cell biology, 132(6): 1133-1149.
[12] Bell J G, Tocher D R, Farndale B M, et al.1997. The effect of dietary lipid on polyunsaturated fatty acid metabolism in atlantic salmon (Salmo salar) undergoing parr-smolt transformation[J]. Lipids, 32(5): 515-525.
[13] Dong C, Weng S, Shi X, et al.2008. Development of a mandarin fish Siniperca chuatsi fry cell line suitable for the study of infectious spleen and kidney necrosis virus (ISKNV)[J]. Virus Research, 135(2): 273-281.
[14] Fryer J L, Lannan C N.1994. Three decades of fish cell culture: A current listing of cell lines derived from fishes[J]. Journal of Tissue Culture Methods, 16(2): 87-94.
[15] Ghosh M C, Ghosh R, Ray A K.2001. Impactof copper on biomonitoring enzyme ethoxyresorufin-o-deethylase in cultured catfish hepatocytes[J]. Environmental Research, 86(2): 167-173.
[16] Hebert P D N, Cywinska A, Ball S L, et al.2003. Biological identification through DNA barcodes[J]. Proceedings of the Royal Society B-biological Sciences, 270: 313-321.
[17] Lai Y S, Murali S, Ju H Y, et al.2010. Two iridovirus-susceptible cell lines established from kidney and liver of grouper, Epinephelus awoara (Temminck & Schlegel), and partial characterization of grouper iridovirus[J]. Journal of Fish Diseases, 23(6): 379-388.
[18] Lakra W, Swaminathan T, Joy K.2011. Development, characterization, conservation and storage of fish cell lines: A review[J]. Fish Physiology and Biochemistry, 37(1): 1-20.
[19] Ryan C M, Carter E A, Jenkins R L, et al.1993. Isolation and long-term culture of human hepatocytes[J]. Surgery, 113(1): 48-54.
[20] Wolf K, Quimby M C.1962. Established eurythermic line of fish cells in vitro[J]. Science, 135(23): 1065-1066.
[21] Wang G, Lapatra S, Zeng L, et al.2004. Establishment, growth, cryopreservation and species of origin identification of three cell lines from white sturgeon, Acipenser transmontanus[J]. Methods in Cell Science, 25(3-4): 211-220.
[22] Ye H Q, Chen S L, Sha Z X, et al.2010. Development and characterization of cell lines from heart, liver, spleen and head kidney of sea perch Lateolabrax japonicus[J]. Journal of Fish Biology, 69(sa): 115-126. |
|
|
|
