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草鱼四倍化细胞株的建立和泥鳅四倍体胚胎的获得
董惠芬
Subtype硕士
Thesis Advisor陈宏溪
1989
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草鱼 泥鳅 电融合 极化注射 四倍体
Abstract本文报导以草鱼肾脏培养细胞系(GCK-84)。大鳞付泥鳅(Paramisgurnus dabryanus),鳗尾泥鳅(Misgurnus anguillicaudatus),白鲫、红鲫及其相互交的囊胚细胞及其未受精卵为材料,运用电融合法和极化注射法,获得草鱼四倍分细胞株和泥鳅四倍体原肠胚等的初步研究结果。1、在本实验室现有条件下,运用正交试验法,分别对草鱼GCK-84细胞系和泥鳅囊胚细胞电融合的最适条件进行研究。其结果是:对于细胞密度为4.2 * 10~5个/ml的GCK-84细胞,预先用40ug/ml的PronaseE在25 ℃下处理10分钟, 在0.2M甘露与8 * 10~(-6)M氯化钙(CaCl_2)配成的电场液中,二核体的比例最高,平均可达40.13%;在这四个因子中细胞密度对二核体比例的影响最为重要,Ca~(2+)浓度的影响也较显著,甘露醇浓度的影响最小。泥鳅囊胚细胞的最佳条件是:在温度为18 ℃时,每毫升2.5 * 10~4个细胞,在0.2M甘露醇为8 * 10~(-5)MCaCl_2配成的电场液中,二核体比例最高可达58.00%,平均为50.99%。这些因子中甘露醇的浓度对实验结果的影响最大,Ca~(2+)浓度的影响最小。2、泥鳅囊胚细胞电融合处理后,随培养时间的延长,二核体中核融合的百分率增加,由1小时的35%增加到6.5小时的84%;而细胞的存活率随时间的延长而下降,由1小时的88%下降到6.5小时的60%。3、运用极化注射法,把融合后的细胞核注射入未受精卵后,能比较正常地发育到囊胚期的卵,从一开始的4%提高到51%,最后提高到88%, 有37.13%的囊胚可继续发育到原肠胚,极少数能孵出鱼苗。对发育到囊胚-原肠期的胚胎,经单个胚胎染色体制片检查证明:染色体数目在90-96条之间;这些染色体加倍了的胚胎占检查胚胎的28%。4、泥鳅的二倍体囊胚细胞核注入未受精卵后所得的鱼,剪尾鳍检查染色体发现:71%以上的分裂相,染色体数目与供体细胞核一样,说明此实验鱼是二倍体鱼。5、在无菌条件下,GCK-84(2n = 48 - 64)融合后进行克隆培养,在54个克隆中筛选出六个四倍化克隆株,染色体分别为96、100、104、108、116、124条,迄今传至第9代。培养细胞的电融合与四倍化细胞的成功筛选,为人工诱导与建立四倍体细胞株提供了另外一种方法。随着短期培养细胞工程鱼囊胚细胞四倍体鱼的成功,培养细胞四倍体工程鱼也会在不久的将来获得成功,这将为人工诱导四倍体鱼开辟一条新途径。
Other AbstractTetraploidized cell strains of grass carp (Ctenopharyngodon idellus) and tetraploid gastrula of loach (Paramisgurnus dabryanus and Misgurnus anguillicaudatus) are obtained from the cultured kidney cell line of grass carp (GCK-84), the blastula cells fused with the unfertilized eggs of loachs using the techniques of electric fusion and polarized injection. 1. The most optimum conditions of the electric fusion of GCK-84 cell line are explored with orthogonal test. The density of cells is 4.2 * 10~5/ml. Cells treated with pronase E(40 ug/ml) for 10 minutes at the temperature 25 ℃. The fusion medium consists of 0.2M mannitol and 8 * 10~(-6)M CaCl_2. Then, the percentage of dikaryon may reach 40.13% on average. Among the above factors, the density of cells is the most important to the percentage of dikaryon, and the next, the concentration of Ca~(2+). The concentration of mannitol is the most unimportant. The most optimum conditions of the fusion of the loach blastula cells are as fallows: The density of cells is 2.5 * 10~4/ml. The temperature is 18 ℃ and the fusion medium consists of 0.2M mannitol and 8 * 10~(-5)M CaCl_2. Then, the percentage or diraryonmay reach 50.99% on average. Among the above tractors, the concentration of mannitol is the most important to the experiment results and the comeootration of Ca~(2+) is the most unimportant. 2. After the blastula cells of loachs was treated with electric fusion, the nuclearfusion rate of dikaryon is increased and the survival rate of the cells is decreased with the passage of time. 3. After the fused nuclei were injected into the unfertilized eggs with the method of polarized injection, 88% eggs can normally develop to blastula stage. Then, 37.13% blastula can reach gastrula stage. Few and far between gastrula can become fry. Chromosome number of the above blastula and gastrula is inspected and 28% is tetraplont. The number of chromosome ineach cell is 90-96. 4. The caudal fin cells of the fish, which were from the unfertilized eggs injected with diploid blastula cell nuclei of loach, were examined. The number of more than 71% metaphase chromosome is the number of diploid chromosome as the same as the donor cell nuclei. 5. In sterility, fused GCK-84 cells were cultured clonally. Six tetraploidized clonal strains were sieved from 54 clones. The numbers of their chromosomes are 96, 100, 108, 116, 124. At present, they have been subcultured to the ninth passages. The successful sieving of tetraploidized cell strain offers another method to induce tetraploid cell strain artificially. And it will also open a new way for inducing tetraploid fish artificially.
Pages52
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12774
Collection学位论文
Recommended Citation
GB/T 7714
董惠芬. 草鱼四倍化细胞株的建立和泥鳅四倍体胚胎的获得[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,1989.
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