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鱼类微细胞和小分离细胞制备技术的研究及其融合初探
徐晓玲
Subtype硕士
Thesis Advisor蒋一珪
1987
Degree Grantor中国科学院中国科学水生生物研究所
Place of Conferral中国科学水生生物研究所
Degree Discipline遗传学
Abstract本文选用方正银鲫(Carassius auratus gibelio)和兴国红鲤(Cyprinus carpio red variety)作为实验材料,运用组织块培养法,建立了银鲤尾鳍组织细胞系CAG-87和兴国红鲤尾鳍组织细胞系CCRV-87。两个细胞系均用TC-199培养基培养。银鲫细胞在PH为6.8,温度为27℃,小牛血清浓度为15%条件下,生长良好,其最高分裂指数可达44%,第22代细胞染色体数为156者占细胞总数的45%。该细胞系已培养了十八个月,传代90次,命名为CAG-87, 属于异倍化的细胞系。红鲤细胞的生长温度,PH值都与前者相同,但小牛血清浓度需要20%。其最高分裂指数为39%, 第24代细胞染色体数为100者占细胞总数90%。此细胞系已培养十七个月,传代85次,命名为CCRV-87。以CCRV-87, CAG-87和CAB-80 (普通鲫鱼囊胚细胞)细胞为材料,对鱼类微细胞和小分离细胞的制备及其形成机制进行了研究。在微细胞的制备中,观察了鱼类细胞微核化过程,微核是由间期状态的细胞核不规则分裂而成;研究了秋水仙胺浓度和处理时间对三种鱼类细胞微核化比例的影响,CAB-80细胞微核化比例较高,可达60%,CCRV-87和CAG-87细胞微核化比例较低为30%;高浓度的秋水仙胺和长时间的药物处理对三种鱼类有毒害作用。小分离细胞聚集体的形成受冷处理时间,温度,PH和培养液等因素的影响。接种16-20小时的CAB-80细胞用浓度 0.3ug/ml 秋水仙胺处理10-14小时,获得有丝分裂中期细胞。在4 ℃低温,PH 8.0条件下处理10小时,小分离细胞聚集体形成的比例可达50%。此外还对小分离细胞聚集体形成的各个阶段进行了光镜,扫描电镜观察,对小分离细胞形成过程中核物质的分配情况进行了观察。结果表明,在小分离细胞形成过程中,细胞核的变化情况与微细胞形成过程中的微核化过程是相同的,但其胞质分裂机制则是不同的。整个细胞最终分裂成为一个小分离细胞聚集体,细胞的微核则随同细胞质的不规则分裂面被分配到各个小分离细胞内。为了探索鱼类遗传育种研究的新途径,把训份染色体转移技术运用于鱼类,我们以小分离细胞为介导转移遗传物质进行了鱼类小分离细胞与受精卵融合,但融时的微核都保持在间期状态,没有与卵核发生融合。
Other AbstractUsing tissue culture technique, we have succesfully established two cell lines from caudal fin of red carp (Cyprinus carpio red variety) and crucian carp (Carassius auratus gibelio), a kind of fish with natural gynogenesis. In TC-199 medium supplemented with 15% calf serum, the cell line of crucian carp, named CAG-87, has been proliferating vigorously through 90 subcultures over a period of 18 month. At the optium gnwth temperature of 27 ℃ and PH value of 6.8, monolayer can be formed about 3-5 days with incubated concentration of 1.2 * 10~4 cells per ml. Twenty-four hours plating, the highest index of mitotic division of cells reaches up to 44%. The precentage of cell with 156 chromosome in 22th passage is 45% and the distribution of chromosome number is ranged from 130-164, so it is heteroploid cell hine. The best growth of red carp cell line requires PH value of 6.8, TC-199 suplemented with 20% calf serum and incubation temperature of 27 ℃. The highest index of mitotci division is 39‰. The precentage of cell with 100 chromosome in 24th passage is 90%. After 85 subcultures over a period of 17 month, the line is named CCRV-87. Using CCRV-87, CAG-87 and CAB-80, a former established fish cell line, we studied some problems about microcells and minisegregant cells (a cluster of small cells which looks like bunches of grapes) such as preparatcon of fish microcells, minisegregant cells and the mechanism of their formation. In the preparation of their formation. In the preparation of mcirocells, the pncedure of micronucleation in fish cells was observed, it showed that the formation of micronuclei was the result from abromal division of interphase nuclei. We also studied the effects of concentration and treatment time of colcemid on three fish cells so that the micromucleation in CAB-80 reaches up 60%, CCRV-87 and CAG-87 reach up 30%. It is harmful to treat fish cells with high concentration or prolonged time of colcemid. The main procedures which can yield large q quantity of fish minisegregant cells are described as follows: 16 to 20 hours after incubation, colcemid is added to cell cultures of CAB-80 at the final concetration of 0.3 ug/ml. Mitotic cells are obtained by continuing in incubation for another 10 to 14 hours. Then the mitotic blocked cells are harvested and stored at 4 ℃ for 10 hours. Mang cells disply highly a bromal pattern of division at PH 8.0 and about 50% of them form minisegregant cells. Analysis of genetic material distribution and observation of electron micronscopy show that during the process of minissegregant cell formattion, the change of nucleu is the same as cell micronueleation, but the cytokinesis are severely disturbed. Micronuclei can be sepreated into m many daughter cells, by irregular cleavage of cytoplasm. In order to explore new route in fish breeding via reconstitution between minisegregant cells and fish fertilized eggs, we introduce the technique of minisegregant cell-mediated chromosome transfer to fish cells and fuse minisegregant cell with fish fertilized eggs. Although minisegregant cells can be fused to fish fertilized eggs with PEG, the fused micronucleis are mantained interphase nuclei and not fused with nuclei of eggs.
Pages60
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12716
Collection学位论文
Recommended Citation
GB/T 7714
徐晓玲. 鱼类微细胞和小分离细胞制备技术的研究及其融合初探[D]. 中国科学水生生物研究所. 中国科学院中国科学水生生物研究所,1987.
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