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Thesis Advisor陈宏溪
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
Abstract本文用Zimmermann的细胞电融合技术。将大鳞付泥鳅(Paramisgurnus dabryanus)的囊胚细胞与同种鱼的未受精卵进行电融合。这种细胞融合卵能够分裂、发育并长成幼鱼和成鱼。细胞事例卵的最初几次卵裂与受畏卵裂不同。球大小不一,排列不规则,有时第一次卵裂就出现2个以上的细胞,细胞数目也可为奇数。但在早期的胚胎发育过程中,这种异常性会逐步地得到校正。至囊胚期,一部分胚胎完全恢复了正常, 可以发育下去直至孵出鱼苗。而没有恢复的异常的胚胎不久就会败育而死亡。本文着重研究了泥鳅囊胚细胞——未受精卵电融合的主要实验条件。其中Ca~(2+)浓度、PronaseE的作用及浓度、不同种类的二价离子,以及甘露醇浓度是影响泥鳅细胞——卵电融合效率的主要理化因素。而供体细胞密度、受体卵质量及其遇清水后被激动的程度是影响融合效率的主要生物因素。通过实验比较得出,在四种二价离子中(Ca~++, Mg~++, Mn~++, Co~++), Ca~++的效果最好,当Ca~++浓度为10~(-3)M、甘露醇浓度为0.2M时,电场液的电导率和渗透压对融合率以及孵化率是最适宜的。用100Mg/ml 的PronaseE在25℃处理供体细胞6~10 min,可以极大地促进融合率。在生物因素中,供体细胞密度在10~5个/ml以下时,融合率随细胞密度的增加而增加,超过10~5个/ml,融合率有下降的趋势,不够成熟的卵或过熟的卵作受体进行电融合,其孵化出苗的可能性一般不大。泥鳅卵迂清水后,被激动程度越高,对其孵化出苗越不利。我们在卵迂水后的10min内完成事例一般都能得到较好的孵化率。通过改善以上几个实验条件,我们实验中的融合率和孵化率分别能够高达80%和20%。 我们用石蜡切片法,对细胞一卵电融合后的早期发育作了细胞学研究。观察到了融进受体卵内的供体细胞核,其数目多为2 ~4 个,位置不定,这些供体细胞核在融合后50min左右,可发育成几个纺体,每个纺缍体作为一个分裂中心,都可引起胞质的分裂, 因此第一次卵裂就形成了多个细胞。细胞融合胚胎的发衣是属于多核还是单核发育,据上前的资料还不卟以作出明确的结论,有待以后的深入研究解决。此外我们还进行了不同属间的电融合,以大鳞付泥鳅(Paramisgurnus dabryanus)的卵为受体,以大鳞付泥鳅 (_T~O)和鳗尾泥鳅(♀)(Misgurnus anguillicaudatus)的杂交的囊胚细胞为供体。在电融合卵发育到原肠中期,检查了它们的染色体。发现它们的数目与供体细胞相同。电融合鱼性状也与供体亲本的性状相同。这些都说明电融合鱼确是由供体细胞核发育而成的。
Other AbstractIn this paper, blastula cells of Paramisgurnus dabryanus were fused with eggs of the same species b by the electrofusion technique. The cell-fused eggs can cleave and develop and finally give birth fires that can grow up to adult fish. The first several cleavages of cell-fused egg are different from that of fertilized egg, blastomeres are disparity in size and irregular in order, some times there are more than two blastomers emerging in the first cleavage, the number of blastomers can be odd. These deformitive embryos can be gradually corrected during the following development, by the time of blastula stage, part of embryos had been corrected to normal and can develop continuously and give birth fries while the abnormal embryos would soon dead. The main experimental conditions of loach's blastula cells-unfertilized eggs electrofution had been studied emphatically in this paper, the results indicated that the concentration of Ca~(2+), mannitol and PronaseE, the types of various divalent cations, the density of donor cells, the quality of recipient eggs and the excitative extent of recipient eggs due to being contacted with water are the primary chemical and biological factors that influence the efficiency of electrofusion. The effect of Ca~(2+) on the electrofusion is the best among the four types of divalent cation: Ca~(2+), Mg~(2+), Mn~(2+), Co~(2+), while the concentration of Ca~(2+)and mannitol are 10~(-3) M and 0.2M respectively, the conductance and osmotic pressure of fusion medium are of best benefit to the rates of fusion and incubation. Pretreated donor cells with 100 Mg/ml PronaseE for 6-10 mim at 25 ℃, th fusion rate is enhanced markedly. Concering the biological factors, fusion rate enhanced markedly with the increasing of donor cells density while the density below 10~5 cells/ml. The possibility of incubation of fry is Little when the quality of recipent eggs is bad. When the loach eggs were contacted with water, the cytoplasm of egg excited immediately, higher the excitative extent, lower the hatching rate, so we accomplished fusion within 10 min after the eggs being contacted with water and obtained the satisfactory hatching rate. By improving the experimental conditions mentioned above, the fusion and hatching rate could reach 80% and 20% respectively in our experiments. Cytological studies on the early development of recipient eggs after cell fusion by the technique of parafin section were carried out. It was observed that after fusion most of the recipient eggs received 2-4 donor cell nuclei which were uncertainly located in the recipient egg cytoplasm. These donor nuclei could develop into cleavage spindles independently 50 min after fusion. Because each spindle acts as a division center and brings about the division of cytoplasm, several blastomeres appeared simutaneously at the first cleavage. It is still not clear; at present whether the development of cell-fused embryos is multinuclear or mononuclear origin, and it is necessary to carry out further investigation to draw conclusion. In additon the intergeneric electrofusions were also carried out with the recipient eggs from Paramisgurnus dabryanus and the donor cells from the hybrid embryos between Paramisgurnus dabryanys () and Misgurnus anguillicandatus (). The chromosome analysis at mid-gastrula showed that the chromosome number is the same as that of donor cells. The characters of the adult electofusion fish are identical with that of donor fish. These results indicate that electrofusion fish does develop from donor cell nucleus.
Document Type学位论文
Recommended Citation
GB/T 7714
田君喜. 泥鳅卵——细胞电融合的研究[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,1988.
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