【作者】 张运海；

【导师】 李宁；

【作者基本信息】 中国农业大学 ， 生物化学与分子生物学， 2005， 博士

【摘要】 猪是人异种器官移植理想的器官来源。体细胞核移植为人们生产不引起免疫排斥以及无内源性病毒的猪提供了可能。本研究系统地研究了猪体细胞克隆相关技术环节,旨在搭建一个能有效生产猪克隆胚的平台。主要内容如下: 利用组织块培养法建立了中国实验用小型猪9个40d胎儿成纤维细胞系和1个成体成纤维细胞系。利用血清饥饿以及接触抑制对细胞进行细胞周期同期化处理,发现细胞同期到G0/G1期的效率相同,而且在处理2d之后,G0/G1期细胞比例不再有显著增加。结果表明胎儿成纤维细胞对血清饥饿以及接触抑制的反应相当,同期处理2d即可为核移植提供足够的供体细胞。 比较了不同培养基,氧分压以及添加胰岛素和LIF对猪卵母细胞体外成熟,成熟后孤雌激活发育的影响,首次尝试将一种新的胚胎培养基PZM-3用于猪卵母细胞体外成熟,还探索了猪体外成熟卵母细胞孤雌激活方案。结果如下:NCSU-23中,卵母细胞核成熟明显优于在TCM199中成熟的效果(p<0.05);1.4kv,100μs,1DC电激活后,卵母细胞死亡率明显高于2.0kv/cm,30μs,1DC和2.0kv/cm,60μs,1DC处理组。但激活后三组间胚胎卵裂率(分别为:72.6%vs.85.9%,73.2%)和囊胚率(分别为:32.1%vs.49.4%,39.0%)相似;化学激活后,胚胎卵裂以及囊胚率都明显不如电激活处理(卵裂率14.4%vs.72.8%;囊胚率3.3%vs.31.5%);以NCSU-23为基础培养基,高氧核成熟显著优于低氧;以改进的PZM-3成熟卵母细胞,核成熟上仍然是高氧优于低氧。若不考虑气相影响,PZM-3的成熟效果明显不如NCSU-23;添加5μg/ml胰岛素后卵母细胞成熟效果显著提高,但是成熟后激活发育没有显著改善。而添加1000 IU/ml LIF卵母细胞核成熟率不但没有明显提高,反而孤雌激活后囊胚率急剧下降,尽管卵裂率以及囊胚细胞总数都没有明显改变。上述结果表明①NCSU23是猪卵母细胞比较理想的培养基,其成熟卵在2.0kv/cm,30μs,1DC或者2.0kv/cm,60μs,1DC电击参数激活下发育比较好;②高氧对卵母细胞核成熟有利,但附植前孤雌胚胎发育具有培养基依赖性;③添加Insulin可以改善卵母细胞核成熟,而添加LIF对卵细胞质成熟有害;④PZM-3如果应用于卵母细胞体外成熟还需要进一步优化。 比较了IVF,PA以及SCNT胚胎的发育率和囊胚质量。三者发育率相似(囊胚率分别为:11.0%,22.6%和16.9%),但是在囊胚质量即ICM/总细胞数比例上,IVF和SCNT胚优于PA胚。比较了PZM-3和NCSU-23培养对孤雌激活胚以及克隆胚发育的影响。对PA胚来讲,PZM-3明显好于NCSU-23(囊胚率35.4%vs.22.6%,p<0.05);而对SCNT胚,PZM-3明显不利于卵裂,但对囊胚发育影响不大(21.1%vs.26.6%,p>0.05)。在NCSU-23+4 mg/ml BSA中添加5μg/ml胰岛素对胚胎发育没有明显促进作用,而添加1000 IU/ml LIF也没发现有损害早期发育的情况。上述结果表明,对孤雌胚或者体外受精胚理想的培养基,当用于克隆胚胎培养时却不一定是最佳的选择;孤雌胚胎在体内发育能力差,可能和ICM/总细胞数很低有关。添加5μg/ml Insulin或者1000IU/ml LIF对孤雌激活胚胎早期发育没有显著影响。 利用脂质体转染技术建立了转GFP的猪胎儿成纤维细胞系,尝试了生产转GFP胚胎的可能性,同时系统研究了供体细胞转染与否、传代次数、准备方式、细胞周期同期化、细胞年龄、细胞形态以及细胞性别等对克隆胚胎早期发育的影响。结果如下:用转GFP的细胞获得克隆胚胎更多还原

【Abstract】 Pigs are considered as ideal organ donor for human future xenotransplantation. Somatic cell nuclear transfer provides an opportunity for producing HAR and endoviruses free pigs. Up to date, there is no somatic cloned pig produced in mainland of our country. So the current research was designed to study main items of pig somatic nuclear transfer systematically such as somatic donors, recipient oocyte, culture of cloned embryos and embryo transfer. It consists of 5 independent experiments.Experiment 1. Nine fetal fibroblast cell lines were set up from 9 fetus of Chinese miniature experimental pigs at 40d of gestation and 1 adult ear skin fibroblast cell lines was set up from fetuses’ mother by using explant-seeding method. Cell cycle stage was synchronized by either serum starvation or contact inhibition. No significant difference of population at G0/G 1 between them and no further increases of G0/G1 cell population after extension of synchronization beyond 2d. It is concluded that fetal fibroblasts responded to serum starvation and contact inhibiton at similar degree and that synchronization for 2d is enough.Experiment 2. Effects of basic maturation media, oxygen tension, insulin and LIF supplementation on nuclear maturation of prepubertal gilts oocytes and subsequent development after parthenogenetic activation were studied. Effect of different activation method on in vitro development of parthenogenetically activated oocytes was also studied. Results are: 1) the rate of nuclear maturation was significant higher after cultured in NCSU-23 than in TCM199; 2) when cultured in NCSU-23, significant higher maturation rate was observed under high oxygen tension （20% O₂） than low oxygen tension （7% O₂）. When cultured in a novel maturation media-PZM, 20% O₂ was better than 7% O₂ also. Regardless of oxygen tension, NCSU-23 supported nuclear maturation better than PZM. 3) Improved maturation rate was obtained after supplementation of insulin, although no significant increase in rates of cleavage and blastocyst after parthenogenetic activation was observed. LIF supplementation did not improve maturation rate. The rate of blastocyst formation decreased drastically after culture in LIF-contaning media although no significant influence on cleavage rate and total cell number of blastocyst were observed. 4) No significant differences among 1.4kv/cm-100μ s-1DC, 2.0kv/cm-30μ s-1DC, 2.0kv/cm-60μs-1DC activation groups as for the rates of cleavage and blastocyst formation. However, 1.4kv/cm-100μs-1DC treatment was significant higher than 2.0kv/cm-30μs-1DC or 2.0kv/cm-60μs-1DC as for death rate of oocytes treated. 5) Chemical activation using Ionomycin and 6-DMAP was poorer than electrical activation using either 2.0kv/cm-30μs-1DC or 2.0kv/cm-60μs-1DC. These results indicated that 1) NCSU-23 is an ideal maturation media and 2.0kv/cm-30μs-1DC or 2.0kv/cm-60μs-1DC electrical activation parameters could effectively activate MII oocytes. 2) High oxygen tension is beneficial to oocytes maturation while preimplatation of PAEs are media dependent. 3) Insulin is beneficial while LIF is detrimental tonuclear maturation of oocytes. 4) A novel media-PZM could be used as maturation media but further studies to optimize it are neededExperiment 3. In the current experiment, in vitro development potential and blastocyst quality of IVFEs, PAEs and NTEs were studied. Preimplantation development competence was similar among IVFEs, PAEs and NTEs. However, with regard to blastocyst quality i.e. ratio of ICM to total cell number, IVFEs and NTEs were superior to PAEs. Effects of culture media including PZM-3 and NCSU-23 on in vitro development potential of PAEs and NTEs were studied. For PAEs, PZM-3 was significant better than NCSU-23. For NTEs, PZM-3 was detrimental to cleavage rate while it had no obvious influence on blastocyst formation. When PAEs cultured in NCSU-23+4 mg/ml BSA supplemented with 5μg/ml Insulin or 1000 IU/ml LIF, no significant improvements of Insulin or no detrimental influence of LIF was observed. Results show that 1) an ide更多还原