【作者】 祝东梅；

【导师】 王卫民；

【作者基本信息】 华中农业大学 ， 水产养殖， 2013， 博士

【摘要】 团头鲂(Megalobrama amblycephala),俗称武昌鱼,隶属于鲤形目,鲤科,鳊亚科,鲂属,原产于我国,起初分布于长江中下游附属的湖泊,自1964年驯化成功后,因其存活率高、生长快、食草性、营养价值高、病害少和易捕捞等优点,现已推广到全国各地养殖。近年来,由于过度捕捞、水域污染和近亲交配,团头鲂种质出现严重退化和混杂现象,养殖群体出现性早熟、生长速度减慢和疾病增多等。目前对团头鲂的研究集中在生物学、免疫、疾病、营养及遗传育种等多个方面,而对团头鲂细胞培养方面的研究报道还很匮乏。因此本研究建立了团头鲂鳍条、肌肉和心脏细胞系,对3种细胞进行鉴定,并将其初步应用于病毒学、环境毒物检测和遗传学等领域。以期将这3种细胞系作为体外体系运用于团头鲂各方面的研究,为团头鲂的养殖和育种提供基础资料；运用于环境毒物检测,以确立敏感合适的细胞体外模型应用于毒物毒性的早期预警研究。具体内容及研究结果如下：(1)团头鲂三种细胞系的建立与鉴定用组织块贴壁法建立了团头鲂鳍条、肌肉和心脏细胞系,在一年多的时间内分别传代到95代、86代和71代以上,分别命名为MAF、MAM和MAH。3-6个月冻存后,MAF、MAM和MAH细胞复苏存活率分别为92.92±5.38%,90.04±5.49%和91.54±6.55%。MAF、MAM和MAH细胞适宜的生长温度为28-32-C,生长培养基为M199,培养基中血清浓度的添加量为10%。第10代的MAF、MAM和MAH细胞染色体数目为48分别占62%、59%和65%,说明3种细胞属正常二倍体细胞。30代MAF、MAM和MAH细胞的12s rRNA片段序列与已报道的团头鲂12srRNA序列一致率为99%-100%,表明3种细胞确实来自于团头鲂。60代MAF、MAM和MAH细胞与细胞角蛋白,纤连蛋白,结蛋白和Ki67抗体均呈阳性反应,表明3种细胞增殖能力强,呈现出上皮样和成纤维样混合状。(2)团头鲂鳍条细胞的初步应用以MAF细胞为研究对象,测定黑鱼棒状病毒(SHRV),鲤春病毒血症病毒(SVCV),斑点叉尾鮰病毒(CCV)和草鱼呼肠孤病毒(GCRV)对MAF细胞的敏感性实验。结果表明,MAF细胞对SHRV, SVCV和CCV较敏感,分别在感染后的1d、2d和2d出现典型的病变；MAF细胞对GCRV不敏感。PCR技术确认了MAF细胞被SHRV、SVCV和CCV感染,而不能被GCRV感染。SHRV、 SVCV和CCV在MAF细胞中病毒滴度分别达到108.2,106.8和105.9TCID50ml-1。MAF细胞对嗜水气单胞菌胞外产物敏感,24h细胞的形态即发生改变,细胞皱缩、变圆,最终整个单层脱落。MAF细胞对重金属铜也很敏感,24h半致死浓度为144.48±13.25μmol/L。用脂质体2000能成功将pEGFP-Nl转入到MAF细胞中,转染效率为5%。上述结果表明MAF作为体外体系能应用于病毒学、细胞生物学和遗传学等领域。(3)全氟辛酸对团头鲂肌肉细胞的毒性研究采用MTT法、细胞总蛋白含量(CP)、中性红摄取试验(NR)法和CCK-8试剂盒测定全氟辛酸对MAM细胞的毒性。实验结果表明：设定的全氟辛酸浓度都对MAM细胞的生长有抑制作用,且细胞存活率的降低与全氟辛酸浓度呈正相关。24h IC50值分别为240.81±10.66mg/L、243.96±11.65mg/L、248.63±13.95mg/L和223.62±13.97mg/L,48h IC50值分别为214.01±10.48mg/L、219.86±10.13mg/L、235.57±8.43mg/L和210.40±8.32mg/L,4种方法测定24h和48h IC50值的大小顺序为NR> CP>MT1>CCK-8,经分析4种方法测定的24h和48h ICso值没有显著性差异(P>0.05)。50mg/L和100mg/L全氟辛酸对MAM细胞暴露染毒后48h,MAM细胞不饱满,有皱缩的趋势,100mg/L组更明显；扫描电镜结果显示：50mg/L全氟辛酸暴露后,MAM细胞核变小,细胞中脂滴数量有增多的趋势,线粒体膨大,100mg/L组MAM细胞中的脂滴数量明显增加,线粒体膨大可以看到明显的嵴。表明全氟辛酸对MAM细胞的线粒体有损伤,并对其脂质代谢有影响。更多还原

【Abstract】 Blunt snout bream (Megalobrama amblycephala), commonly known as Wuchang fish, affiliated to Cypriniformes, Cyprinidae, Abramidinae, Megalobrama. M. amblycephala is native to China, distributed originally to affiliated lakes of middle and lower reaches of the Yangtze River in China. In1964it was domesticated successfully, endowed with excellent biological characteristics for high survival rate, fast growth, herbivorous, high nutritive value, less disease and easy fishing, etc., now it has become one of the main farmed freshwater species in China. In recent years, the germplasm resources of M. amblycephala are under threat of recession and mixture due to overfishing, pollution of waters, inbreeding, and cultured population of M. amblycephala is gradually exhibiting early such as smaller sexual maturity individuals, lose of growth predominance and increase disease. Now, many studies have been conducted to understand its biology, immunology, nutrition, diseases and breeding, but there is limited information available on cultured cells derived from M. amblycephala. Therefore, this study established and characterized three new fish cell lines from the fin, muscle and heart of M. amblycephala, and used these three cell lines as in vitro systems for studying virology, environmental toxicology, genetics and so on. We hope these three cell lines as in vitro systems could be applied in all researches of M. amblycephala, provid basic datas for farming and breeding of M. amblycephala, and identify the more sensitive cell lines as bio-indicators to detect environmental toxic chemicals. The main results were as follows:(1) Development and characterization of three new cell lines from M. amblycephalaThree new fish cell lines (MAF、MAM and MAH) were developed from the fin, muscle and heart of M. amblycephala by using the tissue explants technique, and have been subcultured more than95,86and71times in more than a year. After3-6months of storage in liquid nitrogen, the viability of the MAF, MAM and MAH cells was92.92±5.38%,90.04±5.49%and91.54±6.55%, respectively. All the cell lines were optimally maintained at28-32℃in medium199supplemented with10%fetal bovine serum. Most MAF, MAM and MAH cells have chromosome number of48at passage10, which accounted for62%,59%and65%, respectively, it demonstrated that the three cells are normal diploid cells. DNA was isolated from passage30MAF, MAM and MAH cells, an expected PCR product size was obtained by amplification of12s rRNA from extracted DNA, subsequent comparative analysis of the identified sequences demonstrated a99-100%match with the known mitochondrial DNA sequence of M. amblycephala12S rRNA, the results demonstrated that MAF, MAM and MAH cells indeed originated from M. amblycephala. MAF, MAM and MAH cells were strongly positive for pancytokeratin, desmin, fibronectin and the proliferative marker Ki67, the results demonstrated the three cells to be fibroblastic, epithelial and muscular in nature.(2) The preliminary application of MAF cellsThe susceptibility of MAF cells to infection by four viruses, snakehead rhabdovirus (SHRV), spring viremia carp virus (SVCV), channel catfish virus (CCV) and grass carp reovirus (GCRV), was evaluated by cytopathic effects (CPE). The significant CPE of SHRV, SVCV and CCV infection was observed in MAF cells at1d,2d and2d post-inoculation, respectively. However, no CPE was found in GCRV-infected MAF cells. Furthermore, PCR assays detected prominent bands from SVCV, SHRV and CCV infected MAF cells, it demonstrated MAF cells infected by SVCV, SHRV and CCV. The viral titers of SVCV, SHRV and CCV in MAF cells reached1068,1082and1O5.9TCID50ml-1, respectively. Bacterial cytotoxicity studies showed that extracellular products from Aeromonas hydrophila were toxic to MAF cells, the morphological changes were shrinking, detachment and finally destruction of the monolayer. Cu2+was also cytotoxic to MAF cells and the24h-IC50value was144.48±3.25μmol/L. When MAF cells were transfected with pEGFP-N1plasmid, bright fluorescent signals were observed, and the transfection efficiency reached up to5%. These results suggest that the MAF cell line may provide a valuable tool for studying virus pathogenesis, as well as cytotoxicity testing and genetic manipulation studies.(3) The toxicity studies of perfluorooctanoic acid on MAM cellsCytotoxicity of perfluorooctanoic acid (PFOA) on MAM cells was measured by four endpoint systems:methyl thiazolyl tetrazolium (MTT) assay, cell protein (CP) assay, neutral red (NR) uptake assay and cell counting kit-8(CCK-8) assay. The result showed that PFOA was cytotoxic to MAM cells at all tested concentrations, and toxicity increased as the concentration of PFOA was progressively increased. The24h-IC50values of PFOA were240.81±10.66mg/L,243.96±11.65mg/L,248.63±13.95mg/L and223.62±13.97mg/L for MTT assay, CP assay, NR uptake and CCK-8assay, respectively. The48h-IC50values were214.01±10.48mg/L,219.86±10.13mg/L,235.57±8.43mg/L and210.40±8.32mg/L, respectively. For the four methods,24h-IC50and48h-IC50values were NR>CP>MTT>CCK-8, and there was no significant difference for24h-IC50and48h-IC50values measured by these foure methods (P>0.05). Moreover, the morphological changes of MAM cells were also studied at the concentration of50mg/L and100mg/L for48h. MAM cells were not full, tended to shrinking, and100mg/L group was more obvious. Under a scanning electron microscopy, the nucleus of MAM cells became small, mitochondrial swelled and an increased number of lipid particles after50mg/L PFOA exposure. The number of lipid droplets in MAM cells was significantly increased, and mitochondrial swelled with obvious ridge could be seen in MAM cells at the group of100mg/L. These results indicated PFOA could damage mitochondrial and affect lipid metabolism of MAM cells. This would suggest that the MAM cell line is a suitable bioindicator for the screening of the acute toxicity of PFOA.更多还原