【作者】 王贤丽；

【导师】 樊廷俊； 陈松林；

【作者基本信息】 中国海洋大学 ， 细胞生物学， 2009， 博士

【摘要】 鱼类细胞系的建立始于上世纪60年代,由Wolf和Quimby建立了世界上第一个鱼类细胞系-虹鳟生殖腺细胞系RTG-2。随后,鱼类细胞培养工作取得了较大的进展,一些细胞系相继建立成功。鱼类细胞系因材料容易获得、成本低、重复性好、实验条件可以精确控制等优点,至今已成为一种重要的研究手段,广泛应用于鱼类病毒学等各方面研究。对鱼类细胞系进行深入研究无论在理论研究方面还是在实际应用方面都具有深远意义。本论文研究了半滑舌鳎(Cynoglossus semilaevis)心肌细胞系(HTHC)和圆斑星鲽(Verasper variegates)肾脏细胞系(SHKC)的建立;同时还对半滑舌鳎胚胎细胞系(HEC)和日本牙鲆(Paralichthys olivaceus )胚胎细胞系(FEC)进行了干细胞功能的诱导分化;并将大菱鲆胚胎细胞(TEC)应用于大菱鲆GRIM19基因的功能研究工作上。半滑舌鳎心肌细胞系和圆斑星鲽肾脏细胞培养在添加有20% FBS,1 mM的L-谷氨酸,50 mM的2-ME,100 UI/ml青霉素,100 ug/ml链霉素,10 ng/ml bFGF的MEM培养基中,两种细胞的形态均为成纤维样细胞。HTHC细胞至今已传代30余代;SHKC细胞至今已传代40余代。本实验检测了FBS、bFGF、温度对HTHC和SHKC细胞生长的影响。在一定范围内,细胞生长速度随FBS浓度的增高而加快,FBS浓度过高或过低都抑制细胞的增殖。bFGF对HTHC细胞生长具有刺激作用。HTHC和SHKC细胞在24-30℃之间细胞生长良好,当温度高于30℃或低于12℃时,细胞生长速度降低。HTHC细胞的二倍体核型为2n=42;SHKC细胞的的二倍体核型为2n=46,将EGFP报告基因通过脂质体(lipofectamine2000)介导的方法成功转入HTHC和SHKC细胞中并获得了表达,转化率为10-15%左右。用牙鲆淋巴囊肿病毒(LCVD)感染HTHC和SHKC细胞,观察到了细胞病变效应(CPE),透射电镜检测均在细胞内发现了大量病毒颗粒。半滑舌鳎胚胎细胞系(HEC)和日本牙鲆胚胎细胞系(FEC)为本实验室建立保存。HEC和FEC都具有发育上的多能性,在特定的诱导条件下,都可以诱导分化为纤维细胞、肌肉细胞、神经细胞等各种类型的细胞。全反式视黄酸(RA)、明胶、低浓度FBS均可以诱导两种胚胎细胞分化为纤维细胞;二甲基亚砜和低密度种植HEC细胞均可以诱导HEC细胞分化为树状肌肉细胞;HEC和FEC细胞在低密度条件下均分化为神经细胞、肌肉细胞、上皮细胞;HEC和FEC细胞碱性磷酸酶染色均呈阳性,这些结果初步表明HEC和FEC胚胎细胞具有干细胞的分化特征。将EGFP报告基因通过脂质体(lipofectamine2000)介导的方法成功地转入HEC和FEC中并获得了表达;用牙鲆淋巴囊肿病毒(LCDV)感染HEC和FEC细胞,观察到了细胞病变效应(CPE),通过透射电镜观察到在细胞内有大量病毒颗粒。我们从大菱鲆脾脏cDNA文库中鉴定出一GRIM19基因,并得到该基因的DNA全长。利用RT-PCR技术对该基因进行了在大菱鲆正常组织、不同胚胎发育阶段、鳗弧菌(Vibrio anguillaru,ATCC19019)感染组织和细胞中的表达分析,初步探讨了GRIM19基因在鱼类先天免疫系统中作用。实验结果表明:GRIM19全长cDNA含有一个17 bp的5’UTR,一个435 bp的开放阅读框(open reading frame,ORF)和一个143 bp的3’UTR,开放阅读框编码了144个氨基酸残基。通过比较cDNA序列和基因组序列发现GRIM19基因内含有5个外显子和4个内含子。基因组Southern结果表明该基因为单拷贝基因。系统发生分析表明大菱鲆能够与大西洋庸鲽聚在一起。该基因在正常大菱鲆免疫系统中表达强烈,尤其是在脾脏和头肾中;GRIM19在大菱鲆胚胎不同发育时期表达逐渐增强;在感染头肾、肝脏、脾脏三种组织中,该基因表达均有增加趋势:在感染头肾中该基因表达最强烈,在肝脏感染24 h时基因表达达到最高水平,而在脾脏中该基因表达一直增强;鳗弧菌感染大菱鲆胚胎细胞后,GRIM19基因表达上调。GRIM19基因在濒临死亡的大菱鲆成体鱼肝脏、头肾、心脏和脾脏中的表达均增强。这些结果表明GRIM19基因在大菱鲆免疫应答中起着重要作用。更多还原

【Abstract】 The research of fish cell culture has developed rapidly since Wolf and Quimby established the first fish cell line- RTG-2 in 1960s for the first time. After then, fish cell culture has become an essential research technology which has been used extensively, ranged from virology, environmental toxicology, cytobiology, oncology, genomics, genetics and environmental protection. Cultured fish cells have more advantages than live fish as the experimental material: 1. the materials are cheap and easy to obtain; 2. the experimental condition could be controlled accurately and the experiment could be repeated.In the present study, two cell lines from the heart muscle of Cynoglossus semilaevis and the kidney of Verasper variegates have been established. The induced differentiation of two embryonic cell lines has also been studied. A gene of turbot (Scophthalmus maximus) GRIM19 was screened from a turbot spleen cDNA library. The expression of GRIM19 has been studied using two turbot cell lines.The two cell lines, HTHC and SHKC were cultured in minimum essential medium (MEM) supplemented with fetal bovine serum (FBS) and 10 ng ml-1 basic fibroblast growth factor (bFGF). HTHC cells were subcultured more than 30 times and SHKC cells 40 times. The effects of FBS, bFGF and temperature on the growth of HTHC and SHKC cells had been studied. FBS promotes cell growth at certain concentration and the optimum concentration of FBS was found to be 20% in the medium. bFGF enhanced cell living and propagation ability. The suitable temperature for growth was 20℃to 30℃with the optimum growth at 24℃and a reduced growth at <20℃. The double time of SHKC cells was determined to be 44.8 h. Chromosome analysis revealed that 52% cells maintained normal diploid chromosome number (n=46) in the SHKC cells and 42% in HTHC cells. The HTHC and SHKC cells have been successfully transfected with green fluorescent protein (EGFP) reporter plasmids and the expression of EGFP gene in the cells indicated the possible utility of the cells in gene expression studies. The HTHC and SHKC cells were infected by lymphosystis disease virus (LCDV) and found to be susceptible to the virus by cytopathic effect (CPE) observation. The infection was confirmed by PCR and electron microscopy experiments, which proved the existence of the viral particles in the cytoplasm of the virus-infected cells. In summary, the technology of establishing kidney and heart muscle cell lines is established. It is the first time that HTHC and SHKC were established in the world. Some researches are explored in the application of cell lines and lay foundation of using cell lines to the study in theory and application.Embryonic stem cells are undifferentiated permanent cell lines derived from inner cell mass or primordial germ cells of early developing embryos. In this study, two embryonic cell lines had been induced to study the multifunction of embryonic stem cells. HEC and FEC cells showed pluripotent and differentiation potential in vitro. Under the inducement of all-trans retinoic acid and low concentration of FBS, both of the ES-like cells differentiated into fibroblast-like cells; DMSO induced HEC cells into muscle cells. Under low cell concentration, both of HEC and FEC cells could also be induced into neuron-like cells, fibroblast-like cells, and muscle cells and so on. And FEC cells could also be induced into epidermic cells. Alkaline phosphate activity in HEC and FEC cells were both positive. All the results showed that both HEC and FEC cells were pluripotent embryonic cells. The HEC and FEC cells had been successfully transfected with green fluorescent protein (EGFP) reporter plasmids and the expression of EGFP gene in the cells indicated the possible utility of the cells in gene expression studies. The HEC and FEC cells were infected by lymphosystis disease virus (LCDV) and found to be susceptible to the virus by cytopathic effect (CPE) observation. The infection was confirmed electron microscopy experiments, which proved the existence of the viral particles in the cytoplasm of the virus-infected cells.A turbot (Scophthalmus maximus) gene associated with retinoic/ interferon induced mortality 19 protein-GRIM19 has been screened from a turbot spleen cDNA library. The expression in turbot different tissues, the different embryonic stages had been studied by RT-PCR. The complete cDNA of the turbot GRIM19 contained a 17 bp 5’ UTR, a 435 bp open reading frame (ORF) encoding 144 amino acids and a 143 bp 3’ UTR. Phylogenetic analysis showed that the turbot GRIM19 clustered with Hippoglossus stenolepis. RT-PCR demonstrated that turbot GRIM19 was expressed in spleen and head kidney from uninfected adult. GRIM19 expressed during the early stages of embryo development and gradually increased. The expression of GRIM19 was also dramatically increased after challenge in turbot liver, spleen and head kidney and the expression are most strong in head kidney. The expression reached highest after infection of 24 h in liver and in spleen, the expression was always enhanced. Furthermore, the turbot GRIM19 was induced after 48 h in turbot embryonic cells (TECs) after challenge with Vibrio anguillarum. These results indicated that the turbot GRIM19 played an important role in turbot immune response. Since GRIM19 is a mortality-correlative gene, in the turbot dying tissues infected by Vibrio anguillarum, the GRIM19 expresion was increased in liver, head kidney, heart and spleen. In a word, we report GRIM19 in turbot for the first time. The studies show GRIM19 is an important gene in turbot immunity response process and more research need to be processed for the more GRIM19 functions.更多还原