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题名: 细胞周期同步化研究及其对移核胚胎发育的影响
作者: 刘同明
答辩日期: 2002
导师: 吴清江
专业: 遗传学
授予单位: 中国科学院水生生物研究所
授予地点: 中国科学院水生生物研究所
学位: 博士
关键词: 胚胎细胞 ; 体细胞 ; 细胞周期抑制剂 ; 血清饥饿 ; 同步化 ; 核移植 ; 再程序化 ; 微卫星
其他题名: Studies on Synchronization in Cell Cycle of Cells and Its Effects on the Development of Nuclear Transfer Embryos
摘要: 该论文主要研究了胚胎细胞和培养体细胞的同步化及其对移核胚胎发育的影响.利用细胞生物学和分子生物学方法对未去核卵的异种间核移植模型进行了可行性分析,同时对低温(4℃)短期保存细胞的异种间核移植进行了研究,在此基础上利用RAPD指纹对移核胚胎进行遗传分析.研究内容主要包括以下几个方面:胚胎细胞的同步化研究.主要研究了细胞周期抑制剂噻氨酯甲唑(nocodazole)和秋水仙碱(colchicine)对泥鳅(Misgurnusanguillicaudatus)8-细胞胚胎的同步化效果,同时对两种抑制剂的毒性和可逆性进行了评价.培养细胞的同步化研究.雌核发育鳙(Aristichthysnobilis)尾鳍培养细胞经血清饥饿、低温和4种不同的细胞周期抑制剂处理后,通过流式细胞仪测定细胞DNA含量来分析不同周期细胞和凋亡细胞所占的百分比,同时对血清饥饿后不同大小细胞的细胞周期也进行了分析.影响移核胚胎发育因素的研究.将脱膜后的泥鳅受精卵置于不同孵化液中孵化,结果表明1×Holtfreter中孵化率最高,是最佳孵化液.移核胚胎的遗传分析.以大鳞副泥鳅新鲜分离的囊胚细胞为供体,泥鳅未去核卵为受体进行核移植,利用筛选的14个RAPD引物对供体、移核胚胎和受体进行了遗传分析.
英文摘要: Synchronization in cell cycle of embryonic and somatic cells, and factors affecting the efficiency of nuclear transfer (NT) embryos including synchronized cells were studied. At the same time, nuclear transfer of non-enucleated recipient eggs between different species and donor cells under low temperature for short term were also studied. On this basis, genetic analysis of NT embryos was made by RAPD fingerprinting. Synchronization in cell cycle of embryonic cells. The lowest concentrations of nocodazole and colchicine to arrest and synchronize blastomere division in cleavage-stage loach embryos were determined, and the reversibility and toxicity of treated embryos was assessed. Eight-cell loach embryos were cultured for 4,8,12,or 16hr in 1/lOxHoltfreter supplemented with either nocodazole, an inhibiter of tubulin polymerization, or colchicine, an inhibitor of tubulin assembly. Complete arrest of cell cycle was observed at a nocodazole concentration of 0.275μM, at a colchicine concentration of 0.996mM, respectively. No major morphological alternations in chromatin were observed at the lowest effective concentrations. Reversible and toxic effects of both agents on the development of treated embryos were dose and incubation time dependent. For both agents, prolonging cleavage arrest for more than 4hr (at the effective concentrations) caused embryo lethality. Nocodazole treatment was less cytotoxic, colchicine concentrations which induced cleavage arrest were detrimental to development beyond the blastula stage. Toxic effects beyond the blastula stage could be minimized for both agents by reducing the exposure time of treatment and concentration of cell-cycle-arrest agents. Synchronization in cell cycle of cultured somatic cells. Cultured caudal fin cells from gynogenetic bighead carp were subjected to the following treatments for synchronization, including serum starvation, low temperature and four kinds of cell cycle arrest agents. Distribution of cells in the various phases of the cell cycle and percentages of apoptotic cells were determined by measuring celluar DNA content with flow cytometry. Effects of serum starvation on percentages of cell size and distribution in the various phases of the cell cycle of different-sized cells were also studied. Serum starvation increased percentages of G0+G1 and small-sized cells. At 7th days of serum starvation, percentages of G0+G1 and small-sized cells reached the greatest value (71.46% and 51.42%, respectively), whereas percentages of apoptotic cells reduced to the least value (2.90%). But percentages of small-sized cells in G0+G1 reached the greatest value (97.98%) at 5th days of serum starvation. As the cell size decreased from large to small, percentages of G0+G1 cells increased markedly. Low temperature increased percentages of S and apoptotic cells. Dimethyl sulfoxide(DMSO) increased percentages of G0+G1 cells. Metaphase-arrested agents nocodazole and colchicine increased percentages of G2+M cells. Nocodazole also increased percentages of G0+G1 and apoptotic cells. Colchicine increased abruptly percentages of apoptotic cells from 3.04% of cycling cultures to 60.45% at a concentration of 0.5 pM, but concentrations above 0.5 v- M did not further increased percentages of both G2+M and apoptotic cells. Treatment of 6-dimethylaminopurine (DMAP) simultaneously increased percentages of G2+M and apoptotic cells. Separated cells from flasks were observed when cultures were subjected to low temperature, DMAP and colchicine treatments, it showed that these treatments had considerable detrimental effects on cultured cells. Cultures grew fine when subjected to serum starvation, DMSO and nocodazole treatments, it showed that these treatments may be suitable choices for the cell synchronization for use in studies on effects of donor cell cycle stage on the efficiency of nuclear transfer. Factors affecting the efficiency of somatic nuclear transfer. Fertilized eggs of loach (Misgurnus angitiUicandatus) were cultured in different mediums, the results showed that hatching rate in lx Holtfreter was the highest, so lx Holtfreter is the best incubation medium. For unfertilized eggs of blunt snout bream (Megalobrama ambtycephaia), storage at 15"C for 3 h had no considerable effect on egg quality, but hatching rate of eggs stored for 4 h or 5 h considerably reduced. Mechanic injury had no considerable effects on hatching rate of embryos, but abnormal fries increased considerably (8.8% vs.O). Recipient nuclei of unfertilized eggs from gibel carp (Carasshis auratus gibelio) were enucleated by exposing to 25000 Rads of y-rays. Single nucleus of cultured, synchronized somatic cell from gynogenetic bighead carp (Aristichthys nobilis) was transferred into non-enucleated and enucleated egg of gibel carp, respectively. There was no significant difference in developmental rate between non-enucleated and enucleated recipient eggs (27.27% vs. 25.71%, respectively). Chromosome count showed that 70.59% of NT embryos contained 48 chromosomes. 23.53% of NT embryos consisted of more than 48 chromosomes, it was presumed that those superfluous chromosomes came from recipient eggs. Besides, 5.88% of NT embryos were chimeras. Microsatellite analyses of NT embryos from gibei carp and loach recipient eggs also showed that most of NT embryos came from donor nuclei of bighead carp. Among different duration of serum starvation, developmental rate of NT embryos from somatic nuclei under 3-day serum starvation was the highest, reached 25.71% compared to 14.14%(control), 20%(5-day) and 21.95%(7-day). Eggs of blunt-snout bream (Megalobrama amblycephala) and gibel carp were better recipient eggs than those of loach (Misgunnis angui!Iicaiidatits)(25% and 18.03% vs. 8.43%). There is no considerable difference in developmental rate of NT embryos between loach and large scale loach recipient eggs (7.69% vs.7.02%). Cultured donor cells of less passage facilitated reprogramming of NT embryos than those of more passage. Recloning might improve the developmental rate of NT embryos from the differentiated donor nuclei, especially for the first recloning of somatic cells, developmental rate of late NT blastula increased abruptly. Nuclear transfer of enucleated eggs by exposing to u.v. rays, non-enucleated eggs and stored cells under low temperature for short term. Unfertilized loach eggs exposed to u.v. rays for different time were fertilized by normal sperms. The results showed that nuclei of unfertilized loach eggs were completely inactivated when exposed for 40 s (15 W, 17 cm). Percentage of haploid fries was the highest (63.54%) when unfertilized loach eggs were exposed for 1 min. So 1 min was the optimal time of enucleated nuclei for unfertilized loach eggs by exposing to u.v. rays. Blastula cells of large scale loach were used as donor, enucleated and non-enucleated loach eggs were used as recipient, developmental rate of NT embryos from non-enucleated eggs was higher than that from enucleated eggs. There was no considerable difference in developmental rate of NT embryos from NT blastula cells between non-enucleated and enucleated recipient eggs. For blastula cells of large scale loach, storage at 4 ℃ for 7 d had no considerable effects on shape and viability of cells. Although developmental rate of NT embryos from stored cells under low temperature reduced with the prolonging of storage time, NT fry was obtained, it showed that stored blastula cell under low temperature for short term can be used a donor for nuclear transfer. Stored blastula cells of large scale loach under low temperature for 3 d, freshly blastula cells of large scale loach and freshly blastula cells of loach were used as donor, non-enucleated unfertilized loach eggs were used as recipient, developmental rates of NT blastulae increased in turn (43.96%, 56.65% and 85.39%, respectively). There was no considerable difference in developmental rate of NT embryos between stored blastula cells under low temperature for 3 d and fresh blastula cells of large scale loach in developmental rates of NT embryos at blastopore closure stage, muscular effect stage and fries, respectively. For these three kinds of donors, percentages of NT fries were 1.1%, 0.99% and 1.13%, respectively. Genetic analyis of NT embryos. Blastula cells of large scale loach were used as donor, non-enucleated unfertilized loach eggs were used as recipient, genetic analysis was made among NT embryos, donor and recipient by RAPD fingerprinting of 14 primers. The shared bands by NT embryos and donor were far more than those by NT embryos and recipient (60 vs. 31), genetic distances between NT embryos-donor and NT embryos-recipient were 0.1111 and 0.5634, respectively. It showed that NT embryos came from donor rather than parthenogenesis of recipient, and non-enucleated eggs can be used as recipient for nuclear transfer between different 'species. The shared bands by NT embryos and recipient were a little higher than those by donor and recipient (31 vs. 26), it showed that nuclei of recipient eggs were incompletely rejected and partial DNA fragments of donor nuclei remained in some reconstructed blastulae. There were differences in few bands between NT embryos and donor, some bands disappeared whereas some new bands appeared. It showed that partial genetic materials altered during reprogramming of donor nuclei in recipient eggs, this was responsible for developmental block of some NT embryos.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.ihb.ac.cn/handle/342005/12660
Appears in Collections:中科院水生所知识产出(2009年前)_学位论文

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Recommended Citation:
细胞周期同步化研究及其对移核胚胎发育的影响.刘同明[d].中国科学院水生生物研究所,2002.20-25
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