摘要
近年来,环境污染的日益加剧和各种病毒性疾病的大规模爆发,给鱼类养殖业造成了巨大的经济损失。为查清鱼类病毒的感染途径和感染机理并从根本上解决鱼类病毒病,鱼类细胞系被广泛应用于病毒的分离繁殖、疫苗研制及病毒与宿主细胞的相互作用机理等研究。另外,对海洋中各种环境污染物的检测缺乏快速灵敏有效的毒性检测体系,利用鱼类细胞系可有效地对环境污染物的毒性作用进行评价,确定环境污染物对海洋鱼类产生影响的最低浓度并筛选出合适的生物检测指标,从而与理化检测手段相结合,最终建立快速灵敏有效的环境污染物毒性检测体系。但目前已建立的鱼类细胞系大多来源于淡水和江海洄游性鱼类,其中多数细胞系对海水鱼类病毒不敏感,而海水鱼类细胞系数量不多,可用于鱼类病毒的分离鉴定及体外繁殖及环境污染物的毒性研究及其检测的海水鱼类细胞系更少,远远不能满足对海水鱼类病毒病诊断防治和海洋中各种环境污染物检测的需求。
褐点石斑鱼(Epinephelus fuscoguttatus)隶属于硬骨鱼纲、鲈形目、鮨科、石斑鱼属,是我国重要的海水网箱养殖鱼种之一,同样面临环境污染和病毒性疾病的严重影响,但至今仍未有成功建立褐点石斑鱼组织细胞系的相关报道。因此,迫切需要建立褐点石斑鱼细胞系并应用于鱼类病毒疫苗研制和环境污染物毒性作用评价和检测等方面,为鱼类病毒学和环境毒理学等研究奠定坚实的基础。
为了建立褐点石斑鱼细胞系,本文采用0.5%透明质酸酶和0.2%Ⅱ型胶原酶联合消化法启动了鳍组织的原代培养,采用0.125%胰蛋白酶消化法启动了心脏组织的原代培养,不使用酶消化而仅用组织块培养法启动了鳔组织原代培养。将三种组织培养在24℃,添加有100μg/ mL羧甲基壳寡糖、10ng/mL碱性成纤维样生长因子和40ng/mL I型胰岛素样生长因子的20%胎牛血清(FBS)-DMEM/F-12培养基(pH7.2)中,原代启动7天后分别有细胞迁出,三种细胞均为成纤维样细胞,生长分裂旺盛,22天内三种细胞均长成单层,并能稳定连续传代。细胞生长曲线显示,第60代的褐点石斑鱼鳍、心脏和鳔细胞的群体倍增时间分别为50.6 h,40.3h和43.3h。染色体数目与核型分析显示,第60代褐点石斑鱼三种细胞的特征染色体数目仍为48条,染色体数目和核型特征确定其为褐点石斑鱼细胞。目前,鳍细胞已传至第90代,心脏细胞已传至第70代,鳔细胞已传至第75代,已成功建立褐点石斑鱼鳍、心脏和鳔三种连续性细胞系,将分别利用三种细胞系进行鱼类病毒敏感性和环境污染物毒性作用评价方面的应用研究。
为了研究褐点石斑鱼心脏细胞系对鱼类病毒的敏感性及其体外繁殖病毒的能力,本文利用实验室先前提取并鉴定的两种海水鱼虹彩病毒:大菱鲆红体病虹彩病毒(TRBIV)和淋巴囊肿病毒(LCDV),分别感染褐点石斑鱼心脏细胞系细胞。两种病毒分别加入心脏细胞中吸附2h后,于24℃,10% FBS-DMEM/F12培养液中培养。2-3天后,心脏细胞均出现明显的细胞病变效应(CPE),TRBIV感染的心脏细胞还出现严重的空泡化和颗粒化现象。利用透射电镜可观察到大量典型的TRBIV或LCDV病毒粒子在心脏细胞质中聚集并增殖,病毒粒子大小和形态结构分别与已有报道一致。CPE大于70%后收获心脏细胞后,采用冻融1次,高速冷冻离心的方法得到利用褐点石斑鱼心脏细胞系繁殖的TRBIV或LCDV病毒液。测得TRBIV和LCDV的病毒液滴度较高,分别为104.5 TCID50 ml?1和104.0 TCID50 ml?1,且可继续感染其他正常心脏细胞系细胞。以上结果说明,褐点石斑鱼心脏细胞系对两种鱼类虹彩病毒TRBIV和LCDV较为敏感,在目前的体外病毒繁殖条件下可繁殖获得滴度较高的两种病毒液,褐点石斑鱼心脏细胞系是TRBIV和LCDV良好的体外繁殖体系。
为了利用褐点石斑鱼鳔细胞系研究久效磷对鳔细胞的毒性作用和致毒机理,并对久效磷的毒性作用进行评价以应用于环境中有机磷农药污染检测,本文使用不同浓度久效磷处理褐点石斑鱼鳔细胞系细胞后,发现1μg/mL久效磷即可对鳔细胞产生细胞毒性,且呈浓度依赖性。利用MTT法和细胞蛋白含量测定法所得的久效磷对鳔细胞的48h细胞半数抑制浓度(48h-IC50)分别为30.64和31.44μg/mL。在细胞形态结构方面,久效磷处理鳔细胞72h后,细胞开始出现变圆脱落现象并随后大量死亡。电镜结果显示久效磷主要引起鳔细胞线粒体的严重损伤。随着久效磷浓度的增加,鳔细胞线粒体出现肿胀,内嵴模糊并最终溶解等现象。细胞生物标志酶活性变化结果显示,久效磷可引起鳔细胞乙酰胆碱酯酶(AChE)、超氧化物歧化酶(SOD)和谷胱甘肽-S-转移酶(GST)活性的显著变化。根据细胞病理学及酶学方面的变化,本文推测久效磷的致毒机理是引起细胞内解毒和抗氧化防御系统的损伤,进而影响细胞内线粒体的产能,最终导致细胞死亡。以上结果显示褐点石斑鱼鳔细胞系细胞对久效磷的毒性作用灵敏度高,检测其毒性需要的时间较短,且鳔细胞的AChE、SOD和GST三种酶活性可作为久效磷的生物检测指标,为最终与理化检测手段相结合,建立快速灵敏有效的有机磷污染物生物检测体系奠定基础。
为了利用褐点石斑鱼鳍细胞系对三丁基氧化锡(TBTO)的毒性作用进行评价,研究TBTO对鳍细胞的毒性作用和致毒机理,本文利用不同浓度的TBTO处理褐点石斑鱼鳍细胞系细胞,通过MTT法和细胞蛋白含量测定法检测了TBTO对鳍细胞的体外细胞毒性。由结果可知,1ng/mL TBTO即可对鳍细胞产生细胞毒性,细胞毒性随着TBTO浓度的增加而增大,表现出明显的浓度依赖性。利用上述两种方法所得的TBTO对鳍细胞的48h-IC50值分别为39.87和41.77ng/mL。光镜下观察发现TBTO处理鳍细胞120h内细胞形态和贴壁能力与对照组相比均没有明显可见的改变。而电镜结果则显示TBTO主要引起鳍细胞线粒体的严重损伤,线粒体出现肿胀,内嵴模糊并最终溶解的现象,并在细胞内出现大量空泡。鳍细胞生物标志酶活性变化结果显示,TBTO可引起鳍细胞SOD和GST活性的显著变化,鳍细胞的SOD和GST酶活性可作为TBTO的生物检测指标,为最终与理化检测手段相结合,建立快速灵敏有效且符合国家标准的有机锡污染物生物检测体系奠定基础。
本文旨在建立褐点石斑鱼鳍、心脏和鳔细胞系,并将三种细胞系应用到对鱼类病毒敏感性和环境污染物毒性作用评价的研究中,为鱼类病毒学、环境毒理学及建立快速、灵敏、有效的环境污染物生物评价和检测体系奠定基础。
Fish cell lines are valuable tools for studies of fish virology, cytotoxicology, genetics, and carcinogenesis in vitro. Recently, the degradation of marine environment and outbreak of infectious viral diseases have severely affected many marine fishes and caused great economic losses in aquaculture. Brown-marbled grouper, Epinephelus fuscoguttatus (Forssk?l), is one of the most important commercial fish. And iridovirus have been identified as the most important pathogen of brown-marbled grouper aquaculture, associated with high susceptibility and mortality. To control fish viral diseases, an understanding of the viral dynamics in the host fish cells and in viral vectors is needed. Since fish cell lines can be used as ideal tools for in vitro studies of virus isolation, propagation and cell-virus interactions, it is necessary to develop species-specific cell lines for viral disease investigations. On the other hand, in vitro cultured fish cells have also been found to be rapid and effective evaluation and bioassay systems to screen the cytotoxicity of environmental pollutants.
Many fish cell lines have been established and consist primarily of freshwater and anadromous species. By now, only several cell lines have been established from commercial marine fishes. Unfortunately, no cell line has been established from E. fuscoguttatus. This study was conducted to establish three novel cell lines from E. fuscoguttatus and to characterize their application in the studies of fish viruses and environmental pollutants monitoring.
To establish fin, heart and swim bladder cell lines from E. fuscoguttatus, fin tissues, which were digested with 0.5% hyaluronidase and 0.2% collagenase II; heart tissues, which were digested with 0.125% trypsin; and swim bladder tissues, which were digested without any enzyme, were used respectively to initiate the primary culture in Dulbecco’s Modified Eagle Mediμm: Ham’s Nutrient F-12 (1:1) (DMEM/F12) medium supplemented with fetal bovine serum (FBS), basic fibroblast growth factor, insulin-like growth factor-I and carboxymethyl-chitooligosaccharide at 24 oC. The fin, heart and swim bladder cells in primary culture were all in fibroblastic morphology and proliferated to confluence within 22 days. They grew at a steady rate during subsequent subcultures. Growth property studies indicated that the fin, heart and swim bladder cells had population doubling times of 50.6 h, 40.3h and 43.3h at passage 60, respectively. Karyotype analysis showed that the fin, heart and swim bladder cells all exhibited chromosomal aneuploidy with a modal chromosome nμmber of 48. These implied that the fin, heart and swim bladder cells were indeed brown-marble grouper cells. To date, fin cells have been subcultured up to passage 90, heart cells have been subcultured up to passage 70 and swim bladder cells have been subcultured up to passage 75. They still grow in a good proliferating status. Three continuous brown-marbled grouper fin, heart and swim bladder cell lines which named bmGF-1, bmGH and bmGSB, respectively, have been successfully established.
To evaluate the viral susceptibilities of bmGH cells to two iridoviruses, turbot reddish body iridovirus (TRBIV) and lymphocystis disease virus (LCDV), and in vitro propagation abilities of TRBIV and LCDV utilizing bmGH cells, the bmGH cells at passage 61 were inoculated with TRBIV or LCDV for 2h. Then, viruses were removed and the bmGH cells were cultured in 10% FBS-DMEM/F12 medium at 24 oC. The results of viral susceptibility characterization revealed that typical cytopathic effects (CPE) of bmGH cells emerged after infected by two iridoviruses within 2-3 days. And a large number of TRBIV and LCDV particles were also found in the infected bmGH cells, respectively. The in vitro propagated TRBIV and LCDV had higher infective abilities to reinfect with other normal bmGH cells, when the viruses were harvested after 5-6 days and freeze-thawed for 1 time. The viral titers of TRBIV and LCDV in bmGH cells reached 104.5 TCID50 mL?1 and 104.0 TCID50 mL?1, respectively. All these indicate that bmGH cell line established here may serve as a useful tool for studies of fish virus isolation, propagation and vaccine development.
To establish a rapid and effective bioassay system to detect the organophosphorus insecticide and study their cytotoxic effects and possible toxic mechanisms, bmGSB cells were used to evaluate the acute cytotoxic effects of monocrotophos by MTT assay and cell protein assay. The result showed that monocrotophos was toxic to bmGSB cells at all tested concentrations in concentration-dependent manner. The 48h-IC50 values of monocrotophos to bmGSB cells were 30.64 and 31.44 ug/mL, respectively. After treated by monocrotophos, bmGSB cells became round and lysed at 72h, and began to detach and finally died within 120h. The ultrastructure showed that mitochondria were the most prominent site of monocrotophos cytotoxicity. With increasing monocrotophos concentration, the damage of cell structures was more serious and the mitochondria cristae were totally disrupted and lysed. In the monocrotophos-treated bmGSB cells, the activities of acetylcholinesterase (AChE), superoxide dismutase (SOD) and glutathione S-transferase (GST) changed significantly (P<0.05). It suggested that monocrotophos could affect these enzyme activities with marked changes. The AChE, SOD and GST activities of bmGSB cells could be suitable biomarker for the detection of monocrotophos. All these indicated that bmGSB cell line could be used as a useful cytotoxic evaluation system.
To evaluate and detect the tributyltin oxide (TBTO) and their cytotoxic effects and possible toxic mechanisms, the acute cytotoxic effects of TBTO to bmGF-1 cells were determined by MTT assay and cell protein assay, which indicated the cytotoxicity of TBTO to bmGF-1 cells initiate at 1ng/mL. The 48h-IC50 values of TBTO to bmGF-1 cells were 39.87 and 41.77ng/mL. After treated by TBTO, the bmGF-1 cells exhibit no significant morphological changes as control, but their ultrastructures showed the damage by TBTO. Mitochondria were the most prominent site of TBTO cytotoxicity. With the increase of TBTO concentration, the damage of cell structures became more serious and the mitochondria cristae were totally disrupted and lysed. The result of enzyme activities showed that the activities of SOD and GST in TBTO-treated bmGF-1 cells changed significantly (P<0.05). It suggested that the SOD and GST activities of bmGF-1 cells could be suitable biomarker for the detection of TBTO. All these indicated that bmGF-1 cell line could be used as used a useful cytotoxic evaluation system to detect the TBTO.
This study was conducted to establish three novel cell lines from fin, heart and swim bladder tissues of brown-marbled grouper, and to utilize these cell lines in the study of viral susceptibility evaluation and cytotoxicity detection. These three cell lines can be used as useful tools for studies of fish virology and cytotoxicology, and has potential applications in fish virus isolation, propagation, vaccine development and environmental pollutants monitoring.
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