半滑舌鳎（Cynoglossus semilaevis）细胞培养及细胞系建立研究
摘要
鱼类细胞培养的研究始于20世纪60年代。1962年，Wolf和Quimby建立了世界上第一个鱼类细胞系，即虹鳟鱼生殖腺细胞系RTG-2。此后，鱼类细胞系的建立及其相关研究进展迅速。迄今为止，不仅建立了280余株鱼类细胞系，而且鱼类培养细胞已成为一种重要的研究手段在病毒学、免疫学、鱼类资源保护与遗传育种、肿瘤学、环境毒理学、鱼类生理学、内分泌学等方面的理论及应用研究中发挥作用。因此，对鱼类细胞培养及其细胞系建立进行研究具有深远意义。
半滑舌鳎经济价值高，自然资源量少，是我国新开发的重要海水养殖鱼类。然而，近年来，随着日益加剧的环境污染、养殖规模的扩大以及集约化程度的提高，半滑舌鳎病害导致的死亡事件时有发生，给半滑舌鳎养殖业带来了巨大的经济损失。半滑舌鳎致病病原特性和免疫抗病基因功能的研究是从根本上解决病害的方法；同时，半滑舌鳎雌、雄个体差异非常大，并且属ZW型性别决定体系，是开展性别分化研究的理想材料。细胞系作为一种体外培养系统，因其成本低、试验重复性好、条件可精确控制的优点而成为该鱼种相关研究的重要工具。
本论文选取雌性半滑舌鳎（Cynoglossus semilaevis）头肾、心脏、卵巢组织开展了细胞培养及细胞系建立研究。半滑舌鳎头肾细胞系(TSHKC)、心脏细胞系（TSHC）和卵巢细胞系(TSOC)的培养基为添加20%FBS，50mM2-Me，100U∕ml青霉素，100ug∕ml链霉素，1m M丙酮酸钠，10ng∕ml bFGF的MEM完全培养基，三种细胞的形态均为成纤维样。TSHKC、TSHC、TSOC细胞至今分别已传代40余代，30余代，30代。实验中开展了温度对TSHKC细胞生长影响分析，结果表明TSHKC细胞能在15－30℃温度范围内生长，细胞系的原代培养温度是该细胞系最适宜的生长温度，较高或较低的温度对细胞生长均有抑制作用，其中较高温度对细胞生长的影响最明显。TSHKC、TSHC和TSOC的二倍体核型均为2n=42，且具有异型W染色体。用牙鲆淋巴囊肿（LCDV）分别感染细胞系TSHKC、TSHC和TSOC细胞,观察到细胞病变效应（CPE）,透射电镜下分别在TSHKC、TSHC和TSOC细胞内观察到病毒颗粒。将GFP和GRP报告基因通过脂质体介导的方法转入TSHKC细胞中并成功获得了表达；将GFP报告基因通过脂质体介导的方法分别转入TSHC和TSOC细胞中也成功获得了表达。
文中对雌性半滑舌鳎脾脏、肝脏组织的细胞离体培养进行了初步研究，描述了脾脏组织中淋巴细胞、成纤维样细胞、上皮样细胞的生长特性，对肝脏细胞的离体培养进行了初步探索，为脾脏免疫细胞功能研究奠定基础的同时，为今后脾脏、肝脏细胞系的建立提供了依据。其次，通过脂质体介导的方法将GRP和GFP报告基因分别在脾脏和肝脏的细胞中得到表达，为利用脾脏、肝脏细胞进行外源基因转染及功能研究提供了依据。
综上所述，文中通过不同的鱼类细胞原代培养方法，建立了雌性半滑舌鳎头肾细胞系、心脏细胞系和卵巢细胞系，并从病毒感染性和外源基因转染性两个方面对细胞系的应用性进行研究，为今后在细胞系基础上开展的相关研究奠定了基础。同时，针对雌性半滑舌鳎脾脏、肝脏细胞离体培养及其细胞应用方面的初步研究为脾脏、肝脏细胞在外源基因转染及功能研究中的应用及脾脏、肝脏细胞系的建立提供了依据。
The research of fish cell culture has been developed since1960s. In1962, Wolf and Quimby established the first fish cell line RTG-2in theworld. Subsequently, the establishment of fish cell line and thecorrelational research made progress rapidly. To this day, the number offish cell lines has increased to more than two hundred and eighty,furthermore, the cells in vitro from fishes, as an significant researchtechnology, has played a role in the theory and applied research ofvirology, immunology, fish resources protection, genetic breeding,oncology, environmental toxicology, fish physiology, endocrinology andso on. Therefore, the research on fish cell culture and the establishmentof fish cell lines has the far-reaching significance.
Half-smooth tongue sole (Cynoglossus semilaevis) is an importantaquaculture species in China. However, the diseases with high mortalitiesof half-smooth tongue sole occurred seriously because of the expansivebreeding scale and the intensive aquaculture, it brought huge economicloss to aquaculture. The methods to resolve the diseases fundamentallyare developing the research about the characteristics of the pathogenicagents and the function of disease resistance gene. Meanwhile, thedifference between the male and female individuals in half-smooth tonguesole is big, the fish has the ZW sex-determining system, so half-smoothtongue sole can be used as an ideal material for sex differentiation research. As a kind of in vitro culture system, fish cell lines becomea significant experimental material because of some advantages includinglow cost, high test repeatability and the experimental conditions can becontrolled accurately.
In this paper, the researches about the head kidney, heart, ovarycells of half-smooth tongue sole female individual in vitro culture andcell line establishment have been developed. The fish cell lines, TSHKC,TSHC and TSOC were cultured in minimum essential medium(MEM) supplementedwith fetal bovine serum(FBS), antibiotics,2-Mercaptoethanol (2-Me),sodium pyruvate and basic fibroblast growth factor (bFGF),microscopically, TSHKC,TSHC and TSOC cells are composed offibroblast-like cells. The suitable growth temperature for TSHKC cellswas24℃, Chromosome analysis revealed that43%TSHKC cells maintainednormal diploid number (2n=42), containing the heterogametic W chromosome.The TSHKC cell line was found to susceptible to lymphocystic disease virus(LCDV). The TSHKC cells have been successfully transfected with greenfluorescent protein (GFP) and red fluorescent protein (RFP) reporterplasmids. The growth temperature for TSHC cells was24℃, chromosomeanalysis revealed that52%TSHC cells maintained normal diploid number(2n=42), containing the heterogametic W chromosome. The TSHC cell linewas found to susceptible to lymphocystic disease virus (LCDV). The TSHCcells have been successfully transformed with green fluorescent protein (GFP) reporter plasmids. The growth temperature for TSOC cells was24℃, Chromosome analysis revealed that47%TSOC cells maintained normaldiploid number (2n=42), containing the heterogametic W chromosome. TheTSOC cell line was found to susceptible to lymphocystic disease virus(LCDV). The TSHKC cells have been successfully transformed with greenfluorescent protein (GFP) reporter plasmids.
TO sum up, TSHKC, TSHC,TSOC are established by different primaryculture methods of fish cells, furthermore, the applications of the fishcell lines are studied in two aspects, including virus infection andexogenous gene transfection,it lay foundation for the correlationalresearched that based on these fish cell lines. In addition, the researchabout the spleen and liver cells of half-smooth tongue sole in vitroculture provided the basis not only for the exogenous gene transfectionand functional research with spleen and liver cells, but also for the celllines establishment.
半滑舌鳎经济价值高，自然资源量少，是我国新开发的重要海水养殖鱼类。然而，近年来，随着日益加剧的环境污染、养殖规模的扩大以及集约化程度的提高，半滑舌鳎病害导致的死亡事件时有发生，给半滑舌鳎养殖业带来了巨大的经济损失。半滑舌鳎致病病原特性和免疫抗病基因功能的研究是从根本上解决病害的方法；同时，半滑舌鳎雌、雄个体差异非常大，并且属ZW型性别决定体系，是开展性别分化研究的理想材料。细胞系作为一种体外培养系统，因其成本低、试验重复性好、条件可精确控制的优点而成为该鱼种相关研究的重要工具。
本论文选取雌性半滑舌鳎（Cynoglossus semilaevis）头肾、心脏、卵巢组织开展了细胞培养及细胞系建立研究。半滑舌鳎头肾细胞系(TSHKC)、心脏细胞系（TSHC）和卵巢细胞系(TSOC)的培养基为添加20%FBS，50mM2-Me，100U∕ml青霉素，100ug∕ml链霉素，1m M丙酮酸钠，10ng∕ml bFGF的MEM完全培养基，三种细胞的形态均为成纤维样。TSHKC、TSHC、TSOC细胞至今分别已传代40余代，30余代，30代。实验中开展了温度对TSHKC细胞生长影响分析，结果表明TSHKC细胞能在15－30℃温度范围内生长，细胞系的原代培养温度是该细胞系最适宜的生长温度，较高或较低的温度对细胞生长均有抑制作用，其中较高温度对细胞生长的影响最明显。TSHKC、TSHC和TSOC的二倍体核型均为2n=42，且具有异型W染色体。用牙鲆淋巴囊肿（LCDV）分别感染细胞系TSHKC、TSHC和TSOC细胞,观察到细胞病变效应（CPE）,透射电镜下分别在TSHKC、TSHC和TSOC细胞内观察到病毒颗粒。将GFP和GRP报告基因通过脂质体介导的方法转入TSHKC细胞中并成功获得了表达；将GFP报告基因通过脂质体介导的方法分别转入TSHC和TSOC细胞中也成功获得了表达。
文中对雌性半滑舌鳎脾脏、肝脏组织的细胞离体培养进行了初步研究，描述了脾脏组织中淋巴细胞、成纤维样细胞、上皮样细胞的生长特性，对肝脏细胞的离体培养进行了初步探索，为脾脏免疫细胞功能研究奠定基础的同时，为今后脾脏、肝脏细胞系的建立提供了依据。其次，通过脂质体介导的方法将GRP和GFP报告基因分别在脾脏和肝脏的细胞中得到表达，为利用脾脏、肝脏细胞进行外源基因转染及功能研究提供了依据。
综上所述，文中通过不同的鱼类细胞原代培养方法，建立了雌性半滑舌鳎头肾细胞系、心脏细胞系和卵巢细胞系，并从病毒感染性和外源基因转染性两个方面对细胞系的应用性进行研究，为今后在细胞系基础上开展的相关研究奠定了基础。同时，针对雌性半滑舌鳎脾脏、肝脏细胞离体培养及其细胞应用方面的初步研究为脾脏、肝脏细胞在外源基因转染及功能研究中的应用及脾脏、肝脏细胞系的建立提供了依据。
The research of fish cell culture has been developed since1960s. In1962, Wolf and Quimby established the first fish cell line RTG-2in theworld. Subsequently, the establishment of fish cell line and thecorrelational research made progress rapidly. To this day, the number offish cell lines has increased to more than two hundred and eighty,furthermore, the cells in vitro from fishes, as an significant researchtechnology, has played a role in the theory and applied research ofvirology, immunology, fish resources protection, genetic breeding,oncology, environmental toxicology, fish physiology, endocrinology andso on. Therefore, the research on fish cell culture and the establishmentof fish cell lines has the far-reaching significance.
Half-smooth tongue sole (Cynoglossus semilaevis) is an importantaquaculture species in China. However, the diseases with high mortalitiesof half-smooth tongue sole occurred seriously because of the expansivebreeding scale and the intensive aquaculture, it brought huge economicloss to aquaculture. The methods to resolve the diseases fundamentallyare developing the research about the characteristics of the pathogenicagents and the function of disease resistance gene. Meanwhile, thedifference between the male and female individuals in half-smooth tonguesole is big, the fish has the ZW sex-determining system, so half-smoothtongue sole can be used as an ideal material for sex differentiation research. As a kind of in vitro culture system, fish cell lines becomea significant experimental material because of some advantages includinglow cost, high test repeatability and the experimental conditions can becontrolled accurately.
In this paper, the researches about the head kidney, heart, ovarycells of half-smooth tongue sole female individual in vitro culture andcell line establishment have been developed. The fish cell lines, TSHKC,TSHC and TSOC were cultured in minimum essential medium(MEM) supplementedwith fetal bovine serum(FBS), antibiotics,2-Mercaptoethanol (2-Me),sodium pyruvate and basic fibroblast growth factor (bFGF),microscopically, TSHKC,TSHC and TSOC cells are composed offibroblast-like cells. The suitable growth temperature for TSHKC cellswas24℃, Chromosome analysis revealed that43%TSHKC cells maintainednormal diploid number (2n=42), containing the heterogametic W chromosome.The TSHKC cell line was found to susceptible to lymphocystic disease virus(LCDV). The TSHKC cells have been successfully transfected with greenfluorescent protein (GFP) and red fluorescent protein (RFP) reporterplasmids. The growth temperature for TSHC cells was24℃, chromosomeanalysis revealed that52%TSHC cells maintained normal diploid number(2n=42), containing the heterogametic W chromosome. The TSHC cell linewas found to susceptible to lymphocystic disease virus (LCDV). The TSHCcells have been successfully transformed with green fluorescent protein (GFP) reporter plasmids. The growth temperature for TSOC cells was24℃, Chromosome analysis revealed that47%TSOC cells maintained normaldiploid number (2n=42), containing the heterogametic W chromosome. TheTSOC cell line was found to susceptible to lymphocystic disease virus(LCDV). The TSHKC cells have been successfully transformed with greenfluorescent protein (GFP) reporter plasmids.
TO sum up, TSHKC, TSHC,TSOC are established by different primaryculture methods of fish cells, furthermore, the applications of the fishcell lines are studied in two aspects, including virus infection andexogenous gene transfection,it lay foundation for the correlationalresearched that based on these fish cell lines. In addition, the researchabout the spleen and liver cells of half-smooth tongue sole in vitroculture provided the basis not only for the exogenous gene transfectionand functional research with spleen and liver cells, but also for the celllines establishment.
引文
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