广西卵形鲳鲹小脑来源细胞系的建立及特征分析
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摘要
【目的】卵形鲳鲹(Trachinotus ovatus)是广西重要的海水经济鱼类,但近年来随着养殖规模扩大,各种病害侵染引起的疾病频繁暴发,给广西卵形鲳鲹养殖产业造成重大损失。建立卵形鲳鲹小脑来源的细胞系(Cell line from cerebellum of Trachinotus ovatus,TOCC),将有利于广西卵形鲳鲹养殖中病毒性病害的分离、鉴定和病害的侵染致病分子机理的研究,以及环境污染和变化的动态监测。【方法】采用胰蛋白酶消化法分离得到卵形鲳鲹小脑组织的原代细胞,并进行原代培养。待原代细胞铺满单层后进行传代培养。对稳定传代的TOCC进行18SrDNA基因测序分析,然后进行生长速率分析和染色体分析,并开展病原菌(溶藻弧菌Vibrio alginolyticus)胞外产物对TOCC的毒性分析。【结果】稳定传代的TOCC为成纤维样细胞,18SrDNA基因分析结果显示其来源于卵形鲳鲹;染色体核型分析发现TOCC具有二倍体特征,特征染色体数目为62条;不同培养条件下TOCC细胞的生长速率不同,28℃生长速度最快;病原菌胞外产物对TOCC具有明显的毒性。【结论】本研究成功建立广西卵形鲳鲹小脑组织来源的细胞系(TOCC)。细菌胞外产物对TOCC细胞毒性结果表明,该细胞系在一定范围内能够替代活体检测,实现体外细胞水平对广西海水养殖中病害侵染和环境污染物的快速检测,为养殖病害的有效预防提供技术支持。
【Objective】Golden pompano(Trachinotus ovatus)is a commercially important marine fish species in Guangxi,but in recent years with the expansion of breeding scale,the frequent outbreaks of pathogens caused by various diseases and infections have caused great losses to the aquaculture industry of Trachinotus ovatus in Guangxi.The establishment of a cell line from cerebellum of Trachinotus ovatus(TOCC)would be conducive to the isolation and identification of viral pathogens in Guangxi oval salamander breeding and the pathogenic molecular mechanism of disease infection,as well as the dynamic monitoring of environmental pollution and changes. 【Methods】The primary TOCC cells were cultured by trypsinization,and then subcultured after reaching 100% confluence.18 S rDNA gene sequence analysis was applied to identify the origin of TOCC.We further evaluated TOCC cells' characteristics of growth,chromosome,and toxicity of bacterial extracellular products on TOCC cells.【Results】TOCC cells were fibroblast-like cells,the 18 SrDNA gene sequence analysis showed that it derived from Trachinotus ovatus.Chromosome karyotype analysis found that TOCC had diploid features,the number of characteristic chromosomes was 62.The growth rates of TOCC cell were different in different culture conditions.The optimal culture temperature for TOCC cells growth was 28℃.ECPs exhibited toxicity to TOCC cells.【Conclusion】A novel cell line derived from the cerebellum tissue of Trachinotus ovatus(TOCC)was successfully established in this study.Cytotoxicity results of bacterial extracellular products to TOCC showed that within a certain range the cell line could replace the in vivo detection and realize in vitro cell level rapid detection of the disease infection and environmental pollutants in Guangxi marine culture and provide a technical support for the effective prevention of aquaculture disease.
【Objective】Golden pompano(Trachinotus ovatus)is a commercially important marine fish species in Guangxi,but in recent years with the expansion of breeding scale,the frequent outbreaks of pathogens caused by various diseases and infections have caused great losses to the aquaculture industry of Trachinotus ovatus in Guangxi.The establishment of a cell line from cerebellum of Trachinotus ovatus(TOCC)would be conducive to the isolation and identification of viral pathogens in Guangxi oval salamander breeding and the pathogenic molecular mechanism of disease infection,as well as the dynamic monitoring of environmental pollution and changes. 【Methods】The primary TOCC cells were cultured by trypsinization,and then subcultured after reaching 100% confluence.18 S rDNA gene sequence analysis was applied to identify the origin of TOCC.We further evaluated TOCC cells' characteristics of growth,chromosome,and toxicity of bacterial extracellular products on TOCC cells.【Results】TOCC cells were fibroblast-like cells,the 18 SrDNA gene sequence analysis showed that it derived from Trachinotus ovatus.Chromosome karyotype analysis found that TOCC had diploid features,the number of characteristic chromosomes was 62.The growth rates of TOCC cell were different in different culture conditions.The optimal culture temperature for TOCC cells growth was 28℃.ECPs exhibited toxicity to TOCC cells.【Conclusion】A novel cell line derived from the cerebellum tissue of Trachinotus ovatus(TOCC)was successfully established in this study.Cytotoxicity results of bacterial extracellular products to TOCC showed that within a certain range the cell line could replace the in vivo detection and realize in vitro cell level rapid detection of the disease infection and environmental pollutants in Guangxi marine culture and provide a technical support for the effective prevention of aquaculture disease.
引文
[1]李坚明.“十三五”广西水产养殖业发展战略研究[J].中国渔业经济,2016,34(4):25-31.LI J M.Research on development strategy of Guangxi aquaculture in 13th Five-Year[J].Chinese Fisheries Economics,2016,34(4):25-31.
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[3]张少峰,张春华,邢素坤.广西海洋经济发展现状与对策分析[J].海洋开发与管理,2015(4):103-106.ZHANG S F,ZHANG C H,XING S K.Analysis on the current situation and countermeasures of marine economic development in Guangxi[J].Ocean Development and Management,2015(4):103-106.
[4]夏立群,黄郁葱,鲁义善.卵形鲳鲹主要病害及其研究进展[J].安徽农学通报,2012,18(23):140-143,150.XIA L Q,HUANG Y C,LU Y S.The diseases in Trachinotus ovatus culture and their research progress[J].Anhui Agricultural Science Bulletin,2012,18(23):140-143,150.
[5]崔婧,范雪亭,刘文竹,等.华南地区海水养殖鱼类主要弧菌病原的分离与鉴定[J].海南大学学报:自然科学版,2014,32(3):244-251.CUI J,FAN X T,LIU W Z,et al.Isolation and identification of Vibriosis pathogens of marine cultured fishes in Southern China[J].Natural Science Journal of Hainan University,2014,32(3):244-251.
[6]陈傅晓,唐贤明,谭围,等.卵形鲳鲹深水网箱养殖风险对策分析[J].中国渔业经济,2011,29(4):145-150.CHEN F X,TANG X M,TAN W,et al.The wave-resisted deep-water net-cage breeding of Trachinotus ovatus for risk prevention countermeasures and economic benefit analysis[J].Chinese Fisheries Economics,2011,29(4):145-150.
[7]许海东,区又君,郭志勋,等.神经坏死病毒对卵形鲳鲹的致病性及外壳蛋白基因序列分析[J].上海海洋大学学报,2010,19(4):482-488.XU H D,OU Y J,GUO Z X,et al.The pathogenicity of nervous necrosis virus to Trachinotus ovatus and sequence analysis of the coat protein gene[J].Journal of Shanghai Ocean University,2010,19(4):482-488.
[8]OUYANG Z L,HUANG X H,HUANG E Y,et al.Establishment and characterization of a new marine fish cell line derived from red-spotted grouper Epinephelus akaara[J].Journal of Fish Biology,2010,77(5):1083-1095.
[9]QIN Q W,CHANG S F,NGOH-LIM G H,et al.Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina[J].Dis Aquat Organ,2003,53(1):1-9.
[10]LEE M H,LOH P C.Some properties of an established fish cell line from the marine fish,Caranx mate(Omaka)[J].Proc Soc Exp Biol Med,1975,150(1):40-48.
[11]HUANG X H,HUANG Y H,OUYANG Z L,et al.Establishment of a cell line from the brain of grouper(Epinephelus akaara)for cytotoxicity testing and virus pathogenesis[J].Aquaculture,2011,311:65-73.
[12]LAI Y S,JOHN J A,LIN C H,et al.Establishment of cell lines from a tropical grouper,Epinephelus awoara(Temminck&Schlegel),and their susceptibility to grouper irido-and nodaviruses[J].J Fish Dis,2003,26(1):31-42.
[13]LAI Y S,MURALI S,JU H Y,et al.Two iridovirussusceptible cell lines established front kidney and liver of grouper,Epinephelus awoara(Temminck&Schlegel),and partial characterization of grouper iridovirus[J].Journal of Fish Diseases,2000,23(6):379-388.
[14]蔡小辉,文雪,徐力文,等.卵形鲳鲹一例疑似神经性病毒病的初步分析[J].水产科学,2012,31(10):597-601.CAI X H,WEN X,XU L W,et al.The suspected virus disease diagnose of juvenile ovate pompano Trachinotus ovatus cultured in cages[J].Fisheries Science,2012,31(10):597-601.
[15]周永灿,朱传化,张本,等.卵形鲳鲹大规模死亡的病原及其防治[J].海洋科学,2001,25(4):40-44.ZHOU Y C,ZHU C H,ZHANG B,et al.Isolation and prevention of the pathogen causing large scale death on Trachinotus ovatus[J].Marine Sciences,2001,25(4):40-44.
[16]ZHOU L,LI P,YANG M,et al.Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus(GNNV)with antiviral activities and delivery potential in grouper cells[J].Antiviral Research,2016,129:104-114.
[17]谢从新.鱼类学[M].北京:中国农业出版社,2010:114.XIE C X.Ichthyology[M].Beijing:China Agriculture Press,2010:114.
[18]DONG C F,WENG S P,SHI X J,et al.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,2008,135(2):273-281.
[19]ZHOU L L,LI P F,LIU J X,et al.Establishment and characterization of a mid-kidney cell line derived from golden pompano Trachinotus ovatus,a new cell model for virus pathogenesis and toxicology studies[J].In Vitro Cell Dev Biol Anim,2017,53(4):320-327.
[20]LI P,ZHOU L,WEI S,et al.Establishment and characterization of a cell line from the head kidney of Golden pompano(Trachinotus ovatus),and its application in toxicology and virus susceptibility[J].J Fish Biol,2017,90(5):1944-1959.
[21]HUANG X H,HUANG Y H,SUN J J,et al.Characterization of two grouper Epinephelus akaara cell lines:Application to studies of Singapore grouper iridovirus(SGIV)propagation and virus-host interaction[J].Aquaculture,2009,292:172-179.
[22]YU Y,WEI S,WANG Z,et al.Establishment of a new cell line from the snout tissue of golden pompano Trachinotus ovatus,and its application in virus susceptibility[J].J Fish Bio,2016,88(6):2251-2262.
[23]赖迎迢,陶家发,孙承文,等.鱼源溶藻弧菌生物学特性和病理组织学观察[J].微生物学报,2014,54(11):1378-1384.LAI Y T,TAO J F,SUN C W,et al.Biological characteristics and histopathological observation of Vibrio alginolyticus from diseased fish[J].Acta Microbiologica Sinica,2014,54(11):1378-1384.
[24]LI P F,YU Q,ZHOU L L,et al.Probing and characterizing the high specific sequences of ssDNA aptamer against SGIV-infected cells[J].Virus Research,2018,246:46-54.
[25]LI P F,ZHOU L L,WEI J G,et al.Development and characterization of aptamer-based enzyme-linked aptasorbent assay for the detection of Singapore grouper iridovirus infection[J].J Appl Microbiol,2016,121(3):634-643.
[26]ZHOU L,LI P,NI S,et al.Rapid and sensitive detection of redspotted grouper nervous necrosis virus(RGNNV)infection by aptamer-coat protein-aptamer sandwich enzyme-linked apta-sorbent assay(ELASA)[J].J Fish Dis,2017,40(12):1831-1838.
[27]LI P,WEI S,ZHOU L,et al.Selection and characterization of novel DNA aptamers specifically recognized by Singapore grouper iridovirus-infected fish cells[J].J Gen Virol,2015,96:3348-3359.
[2]刘锡强,马学坤,刘康,等.华南地区金鲳鱼养殖报告[J].当代水产,2014(2):26-29.LIU X Q,MA X K,LIU K,et al.Reports on the cultured Trachinotus ovatus in South China[J].Current Fisheries,2014(2):26-29.
[3]张少峰,张春华,邢素坤.广西海洋经济发展现状与对策分析[J].海洋开发与管理,2015(4):103-106.ZHANG S F,ZHANG C H,XING S K.Analysis on the current situation and countermeasures of marine economic development in Guangxi[J].Ocean Development and Management,2015(4):103-106.
[4]夏立群,黄郁葱,鲁义善.卵形鲳鲹主要病害及其研究进展[J].安徽农学通报,2012,18(23):140-143,150.XIA L Q,HUANG Y C,LU Y S.The diseases in Trachinotus ovatus culture and their research progress[J].Anhui Agricultural Science Bulletin,2012,18(23):140-143,150.
[5]崔婧,范雪亭,刘文竹,等.华南地区海水养殖鱼类主要弧菌病原的分离与鉴定[J].海南大学学报:自然科学版,2014,32(3):244-251.CUI J,FAN X T,LIU W Z,et al.Isolation and identification of Vibriosis pathogens of marine cultured fishes in Southern China[J].Natural Science Journal of Hainan University,2014,32(3):244-251.
[6]陈傅晓,唐贤明,谭围,等.卵形鲳鲹深水网箱养殖风险对策分析[J].中国渔业经济,2011,29(4):145-150.CHEN F X,TANG X M,TAN W,et al.The wave-resisted deep-water net-cage breeding of Trachinotus ovatus for risk prevention countermeasures and economic benefit analysis[J].Chinese Fisheries Economics,2011,29(4):145-150.
[7]许海东,区又君,郭志勋,等.神经坏死病毒对卵形鲳鲹的致病性及外壳蛋白基因序列分析[J].上海海洋大学学报,2010,19(4):482-488.XU H D,OU Y J,GUO Z X,et al.The pathogenicity of nervous necrosis virus to Trachinotus ovatus and sequence analysis of the coat protein gene[J].Journal of Shanghai Ocean University,2010,19(4):482-488.
[8]OUYANG Z L,HUANG X H,HUANG E Y,et al.Establishment and characterization of a new marine fish cell line derived from red-spotted grouper Epinephelus akaara[J].Journal of Fish Biology,2010,77(5):1083-1095.
[9]QIN Q W,CHANG S F,NGOH-LIM G H,et al.Characterization of a novel ranavirus isolated from grouper Epinephelus tauvina[J].Dis Aquat Organ,2003,53(1):1-9.
[10]LEE M H,LOH P C.Some properties of an established fish cell line from the marine fish,Caranx mate(Omaka)[J].Proc Soc Exp Biol Med,1975,150(1):40-48.
[11]HUANG X H,HUANG Y H,OUYANG Z L,et al.Establishment of a cell line from the brain of grouper(Epinephelus akaara)for cytotoxicity testing and virus pathogenesis[J].Aquaculture,2011,311:65-73.
[12]LAI Y S,JOHN J A,LIN C H,et al.Establishment of cell lines from a tropical grouper,Epinephelus awoara(Temminck&Schlegel),and their susceptibility to grouper irido-and nodaviruses[J].J Fish Dis,2003,26(1):31-42.
[13]LAI Y S,MURALI S,JU H Y,et al.Two iridovirussusceptible cell lines established front kidney and liver of grouper,Epinephelus awoara(Temminck&Schlegel),and partial characterization of grouper iridovirus[J].Journal of Fish Diseases,2000,23(6):379-388.
[14]蔡小辉,文雪,徐力文,等.卵形鲳鲹一例疑似神经性病毒病的初步分析[J].水产科学,2012,31(10):597-601.CAI X H,WEN X,XU L W,et al.The suspected virus disease diagnose of juvenile ovate pompano Trachinotus ovatus cultured in cages[J].Fisheries Science,2012,31(10):597-601.
[15]周永灿,朱传化,张本,等.卵形鲳鲹大规模死亡的病原及其防治[J].海洋科学,2001,25(4):40-44.ZHOU Y C,ZHU C H,ZHANG B,et al.Isolation and prevention of the pathogen causing large scale death on Trachinotus ovatus[J].Marine Sciences,2001,25(4):40-44.
[16]ZHOU L,LI P,YANG M,et al.Generation and characterization of novel DNA aptamers against coat protein of grouper nervous necrosis virus(GNNV)with antiviral activities and delivery potential in grouper cells[J].Antiviral Research,2016,129:104-114.
[17]谢从新.鱼类学[M].北京:中国农业出版社,2010:114.XIE C X.Ichthyology[M].Beijing:China Agriculture Press,2010:114.
[18]DONG C F,WENG S P,SHI X J,et al.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,2008,135(2):273-281.
[19]ZHOU L L,LI P F,LIU J X,et al.Establishment and characterization of a mid-kidney cell line derived from golden pompano Trachinotus ovatus,a new cell model for virus pathogenesis and toxicology studies[J].In Vitro Cell Dev Biol Anim,2017,53(4):320-327.
[20]LI P,ZHOU L,WEI S,et al.Establishment and characterization of a cell line from the head kidney of Golden pompano(Trachinotus ovatus),and its application in toxicology and virus susceptibility[J].J Fish Biol,2017,90(5):1944-1959.
[21]HUANG X H,HUANG Y H,SUN J J,et al.Characterization of two grouper Epinephelus akaara cell lines:Application to studies of Singapore grouper iridovirus(SGIV)propagation and virus-host interaction[J].Aquaculture,2009,292:172-179.
[22]YU Y,WEI S,WANG Z,et al.Establishment of a new cell line from the snout tissue of golden pompano Trachinotus ovatus,and its application in virus susceptibility[J].J Fish Bio,2016,88(6):2251-2262.
[23]赖迎迢,陶家发,孙承文,等.鱼源溶藻弧菌生物学特性和病理组织学观察[J].微生物学报,2014,54(11):1378-1384.LAI Y T,TAO J F,SUN C W,et al.Biological characteristics and histopathological observation of Vibrio alginolyticus from diseased fish[J].Acta Microbiologica Sinica,2014,54(11):1378-1384.
[24]LI P F,YU Q,ZHOU L L,et al.Probing and characterizing the high specific sequences of ssDNA aptamer against SGIV-infected cells[J].Virus Research,2018,246:46-54.
[25]LI P F,ZHOU L L,WEI J G,et al.Development and characterization of aptamer-based enzyme-linked aptasorbent assay for the detection of Singapore grouper iridovirus infection[J].J Appl Microbiol,2016,121(3):634-643.
[26]ZHOU L,LI P,NI S,et al.Rapid and sensitive detection of redspotted grouper nervous necrosis virus(RGNNV)infection by aptamer-coat protein-aptamer sandwich enzyme-linked apta-sorbent assay(ELASA)[J].J Fish Dis,2017,40(12):1831-1838.
[27]LI P,WEI S,ZHOU L,et al.Selection and characterization of novel DNA aptamers specifically recognized by Singapore grouper iridovirus-infected fish cells[J].J Gen Virol,2015,96:3348-3359.