| 题名 | 猕猴胚胎干细胞神经分化及猕猴胚胎移植时间窗口研究 |
| 作者 | 陈永昌 |
| 学位类别 | 博士 |
| 答辩日期 | 2010-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 季维智 |
| 关键词 | 猕猴胚胎干细胞 神经分化 时间窗口 |
| 其他题名 | Neural Differentiation of Rhesus Monkey Embryonic Stem Cells and Time Window for Embryo Transfer in Rhesus Monkey |
| 学位专业 | 动物学 |
| 中文摘要 | 猕猴在解剖学、遗传学和生理学上与人类极为接近，是最常用和最重要的灵长类实验动物。灵长类中最早被建系的猕猴胚胎干细胞（rESCs）已经成为重要的模型材料，被广泛应用于胚胎早期发育体外研究和退行性疾病的细胞替代治疗研究等领域。足够数量卵子的获得是开展胚胎生物学研究的最基本过程，而胚胎移植则是实现人为控制下诞生目标个体的重要途径。本文对灵长类ESCs的神经分化及灵长类辅助生殖技术研究进展进行了综述，并对以下几方面开展了研究：利用rESCs向神经分化的过程，研究了p53及其相关蛋白在早期神经分化中的表达变化情况；以rESCs为模型，通过调控分化培养体系中的叶酸浓度初步研究了叶酸对神经分化的作用；以时间窗口为出发点，详细分析了猕猴超数排卵及胚胎移植过程中若干时间窗口对卵子的产出以及胚胎妊娠的重要影响。主要试验结果如下：1）rESCs在神经诱导微环境下，FGF-2为主的生长因子使细胞增殖速度加快，并诱导ESCs分化为更高比例的神经前体细胞，同时引起ASPP家族表达变化进而调节p53表达上调，p53一方面抑制Oct-4等的表达而使ESCs分化，另一方面启动原凋亡蛋白Bax和PIG-3表达上调，导致神经分化过程凋亡的发生也更加明显。2）诱导rESCs向神经分化过程中，叶酸缺乏可引起细胞增殖减缓、神经前体细胞rosettes结构减少但并不影响ESCs转化为神经前体细胞的效率。然而神经前体细胞进一步分化结果显示，叶酸缺乏不影响胶质细胞的分化，但影响神经元的分化。叶酸拮抗剂MTX影响细胞的增殖、神经前体细胞的诱导和神经元的分化，添加过量叶酸可减缓MTX对细胞增殖和分化的影响。3）猕猴年龄、超排间隔和生殖季节等时间窗口对超排取卵结果起着重要作用，我们将2-8细胞到囊胚阶段的胚胎通过输卵管移植，妊娠结果显示，卵泡期晚期到黄体期早期是猕猴胚胎移植的时间窗口。 本论文首次对p53在灵长类ESCs神经分化过程中的作用开展研究，并初步探索了叶酸对神经分化的作用。这些研究有望在体外深入探索早期胚胎正常或异常神经发育的机理问题。胚胎移植时间窗口的发现对提高猕猴胚胎移植后妊娠率具有重要意义，可以作为今后开展此类试验的参考依据。 |
| 英文摘要 | The rhesus monkey has been one of the most widely used laboratory animal in medical and biological research due to the close relationship to human being in anatomy, genetics and physiology. Rhesus monkey embryonic stem cell (rESC) is the ideal model for study on embryologic developmental biology and cell replacement therapy for human cellular degenerative diseases. Meanwhile, to obtain sufficient oocytes by superovulation (SO) is essential process for embryonic biotechnology, and embryo transfer (ET) is an important way to produce offspring from in vitro produced embryos. In this thesis, the progress in neural differenatioation from primate ESCs and assisted reproductive technology (ART) in primate were reviewed. Furthermore, experiments focused on neural differentiation from rESCs and the time windows during SO-ET in rhesus monkeys were designed and ferformed, which including: 1) The p53 and its regulator ASPP family, p53 targets Bax, PIG-3 and p21waf1 were detected during the process of neural differentiation from rESCs. The results showed that FGF-2 and other neural induction factors promote cell proliferation, neural conversion, and apoptosis during the differentiation process.The ASPP family members changed their expression either in quantity or pattern to regulate p53 in this process. The p53 actived its proapoptotic targets Bax and PIG-3 but not cell cycle arrest target p21waf1. The results implied that neural differentiation is concomitant with apoptosis in rESCs. 2) The roles of folic acid (FA) during neural differentiation from rESCs were assayed by regulating FA concentration or introducing the antagonist of FA: methotrexate (MTX). Our data showed that FA deficiency decreased cell proliferation and reduced the formation of neural rosettes. No obvious difference in expression of nestin and Pax6 (neural progenitor markers) among neural progenitors obtained from all FA concentration groups was observed. However, FA deficiency affected neuronal differentistion, but not neuroglia. Furtherly, MTX reduced cell expansion, neural induction and neuronal differnatiation, and excessive FA supply was able to partly rescue MTX’s effects. 3）The results of SO and ET in rhesus monkeys were summarized and the reasons that could result in SO-ET failure were analyzed. We found that the time windows such as age stages, repeated stimulating with recombinant human gonadotropins and restimulation interval, and breeding season were the key factors for SO-ET. Importantly, 2-8-cell embryos to blastocysts were transferred through oviduct embryo transfer. We found that the optimal time window for embryo transfer in rhesus monkeys might be the stage from late follicular phase to early luteal phase. For the first time, we found some novel roles of p53 and folic acid during the rESCs neural differentiation. These preliminary data were expected to be helpful for learning correlative mechanism of normal or abnormal development in nervous system. And the finding of time window for embryo transfer might be an important reference to further work. |
| 语种 | 中文 |
| 公开日期 | 2013-04-24 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7399]  |
| 专题 | 昆明动物研究所_生殖与发育生物学 |
| 推荐引用方式 GB/T 7714 | 陈永昌. 猕猴胚胎干细胞神经分化及猕猴胚胎移植时间窗口研究[D]. 北京. 中国科学院研究生院. 2010. |
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