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中华鲟Acipenser sinensis吻端和性腺细胞系的生物学特性
Thesis Advisor刘汉勤
Degree Grantor中国科学院水生生物研究所
Place of Conferral中国科学院水生生物研究所
Degree Discipline水生生物学
Keyword中华鲟acipenser Sinensis 组织培养 生物学特性 染色体
Abstract本篇硕士论文可分为两部分:前半部分为综述部分,总结了鲟鱼类的资源概况以及鲟鱼类体细胞和分子遗传学研究的概况,并对体细胞遗传学领域若干重要技术的应用情况进行了文献综述。后半部分是实验部分,以中华鲟Acipenser sinensis为材料进行了组织培养,并对所建立的细胞系的部分生物学特性进行了鉴定,还尝试用RAPD技术检测不同代数培养细胞相对于来源个体的遗传相似性和变异程度。实验中,在无菌条件下分别取1龄中华鲟的尾鳍、吻端、皮肤、肌肉、肝脏、肠、性腺、肾等八种组织,采用组织块贴壁法,使用含20%的小牛血清的TC199培养基,在27℃下进行体外原代培养,并在传代培养过程中逐渐降低血清浓度至10%。得到来源于吻端和性腺组织的两个细胞系,分别命名为CSSn和CSG。CSSn和CSG均以成纤维样细胞为主。细胞生长较慢,CGS接种9天后、CSSn接种14天后数目才达到最高值,并且数量仅为原来接种时的2倍左右。用草鱼出血病病毒GCHV和甲鱼虹彩病毒STIV感染后,细胞大量脱落和死亡。生长温度范围和最适pH的实验表明,CSG在16~30℃生长正常,最佳生长温度在27℃附近,最适pH为7.2。对第9代CSG和第31代CSSn的染色体进行了分析。CSSn和CSG均含有微染色体(含有微染色体是鲟形目染色体的重要特点)。这两株中华鲟培养细胞的染色体数目众多,在分布上的偏离正常2n值,数目变动范围很大,呈现出“双峰多态”的特点:数目分布范围广,众数区间为两个。第9代CSG的染色体分布的中心趋向落在222~234和248~260之间,各占计数细胞总数的20.2%。第31代CSSn在196~208区间内出现率最高,为23.5%;其次是在170~182区间,出现率15.3%。研究显示这两种培养细胞己属异倍体。大多数细胞染色体数低于正常2n值(264)。提取第13、25、28和34代CSG和DNA以及提供该组织的中华鲟的肌肉DNA,用RAPD技术进行相似性分析。通过统计分析发现随着在体外培养时间的增长,细胞DNA变异程度加大,与来源个体的DNA相似性降低,提示体外环境具有一定的选择压力。结合染色体数目分析来看,染色体丢失和DNA水平上的变异,表征了细胞的异倍化进程,使细胞向着适应体外培养环境的方向转化。
Other AbstractTwo main parts are included in present thesis: The former is a part of reviews. A general situation of resources of Acipenseriformes as well as their somatic cells nd molecular genetic research is given. A review of the research progress and application of several sorts of technology in the fields of somatic genetics is also accomplished. The latter is the one about my experiments. The Chinese sturgeon Acipenser sinensis was used as material for tissue culture. Biological characteristics of established Chinese sturgeon cell lines were partly examined. RAPD were studied to detect the heredity similarity and variety of cultured cells at different passage, as well as in compare with the original individual. In the experiments, eight tissues including tail fin, snout, skin, muscle, liver, Intestine, gonad and kidney were aseptically removed from a yearling Chinese sturgeon, and minced in separate dishes containing TC199 medium and 20% fetal calf serum buffered with N-2-hydroxyethylpiperazine-N-2-ethane-sulfonicacid (HEPES), and incubated at 27℃ for in vitro primary culture. The concentration of serum was gradually reduced to 10% during the subculture. Two cell lines were established from snout and gonad tissues, and designated as CSSn and CSG, respectively. Both CSSn and CSG appeared fibroblastic. Cultured cells grew very slowly. After seeding for 9 and 14 days, respectively, CGS and CSSn got the highest number, as well as the growth quantity was only about double of seeding amount. After infected by Grass Carp Hemorrhage Virus GCHV and Soft-shelled turtle iridovirus STIV, cells divorced from dishes to death. The effect of temperature and pH on growth were examined for CSG, which remained a normal state at 16~30℃. Optimum temperature was 27℃ or so, while a proper pH condition is 7.2. The chromosomes of CSG at passage 9 and CSSn at passage 31 were analyzed. Both cell lines possessed microchromosome, which was an important character of Acipenseriformes chromosomes. These two lines had large quantity of chromosomes, away from normal 2n number in distribution, which exhibited a same feature described as "two wave crests distinguished and a great variety of distribution": two mass districts and a large scale distribution. The central trend of CSG at passage 9 was in scale of 222~234 and 248~260, which occupied 20.2% of the total, respectively. CSSn at passage 31 had the highest appearance, 23.5% in scale of 196~208, and the secondarily highest, 15.3% in 170~182. The investigation demonstrated that the two lines became aneuploidy. Different from general fish cells lines, sturgeon cells lost their chromosomes in large quantities, thus quantity of chromosomes in the majority were lower than 264, the normal 2n number. DNA of CSG at passage 13、25、28、34 and muscle of original individual were drawed for similarity analysis with RAPD technology. Through static analysis, its found that as the time of in vitro culture went by, the variety of DNA was enlarged and the heredity similarity compared with the original individual was reduced, which suggested outer circumstance kept certain selection pressure. Combined with chromosomes analysis, the loss of chromosome and the variety of DNA level suggested the aneuploidy course, which would lead cells to adaptability to in vitro circumstances.
Document Type学位论文
Recommended Citation
GB/T 7714
叶湘辉. 中华鲟Acipenser sinensis吻端和性腺细胞系的生物学特性[D]. 中国科学院水生生物研究所. 中国科学院水生生物研究所,2000.
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