猕猴体细胞核移植影响因素的研究：着床前胚胎表观遗传重编程和供体细胞周期同步化
文献类型：学位论文
| 作者 | 杨纪峰 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-06 |
| 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 | 猕猴着床前胚胎发育 体细胞核移植 表观遗传重编程 DNA 甲基化 细胞周期同步化处理 |
| 其他题名 | Studies on monkey cloning: Epigenetic marks in early embryos development and treatment on somatic cells before nuclear transfer |
| 学位专业 | 动物学 |
| 中文摘要 | 体细胞核移植（somatic cell nuclear transfer）克隆技术的成功，特别是运用终末分化的淋巴细胞和嗅觉神经元细胞成功克隆出小鼠，证实了分化的体细胞核潜在的发育全能性。该技术已经在多个物种上成功地得到克隆后代，在转基因动物、基因敲除动物和疾病模型动物生产中也得到成功应用，在结合干细胞技术的治疗性克隆和再生医学方面也取得了初步成果，展现出了具有深远意义的应用前景。但是，目前该领域仍然存在着很多急待解决的重要问题：克隆成功率低，克隆胚和克隆动物经常呈现发育异常，妊娠和出生前后的高死亡率。对哺乳动物早期胚胎发育过程中DNA甲基化、组蛋白修饰等表观遗传重编程（epigenetic reprogramming）机制的深入了解，有助于研究体细胞核在去核卵母细胞中的表观遗传重编程事件，进而改善克隆胚重编程效率和发育能力。 猕猴是一种重要的实验动物，在人类疾病模型和生物医药研究中有重要的意义。本研究主要围绕猕猴体细胞克隆胚胎早期发育过程中的表观遗传重编程事件和核移植前体细胞同步化处理这两方面展开。1），首次详细地了描绘了猕猴着床前胚胎发育过程中整体水平的DNA甲基化表观遗传重编程事件，研究发现在受精卵中父本基因组形成原核后迅速地发生了去甲基化，在2细胞期后的卵裂过程中，母本基因组才开始逐渐地去甲基化，到桑葚胚达到最低水平，然后开始重新（de novo）甲基化,到囊胚期时形成不对称的甲基化模式，滋养外胚层（TE）呈现高甲基化状态，而内细胞团（ICM）呈现低甲基化状态,这一不对称模式可能是灵长类动物特有的，其他哺乳动物呈现正好相反的不对称模式。2），研究发现，大多数猕猴克隆胚胎的DNA甲基化重编程存在异常,效率低。很多2细胞期克隆胚（67%）和8细胞期克隆胚（50%）的核DNA甲基化水平显著高于对应的体外受精胚，8细胞克隆胚之间呈现多种不同的表观遗传特征。大多数克隆囊胚的ICM细胞核的甲基化水平显著高于IVF囊胚，这些异常可能是导致克隆胚胎移植到代孕母体后发育时间不长就失败的原因。3），在核移植前对猕猴成纤维细胞同步化处理的研究中发现，血清饥饿，细胞周期阻断剂DMSO（二甲基亚砜）、roscovitine、aphidicolin和indirubin的处理都有显著的同步化效果，提高了G0+G1期细胞的比例。经过BrdU标记法证实了这几种处理方法抑制细胞增殖的效果，并且证实了这种周期阻滞作用是可逆的。用原位末端标记法（TUNEL）分析证实，血清饥饿1到4天后细胞凋亡比例显著上升，在贴壁的细胞中约有6%发生凋亡，而正常对照只有1%左右，而周期阻断剂处理没有增加细胞凋亡率，这提示这些周期阻断剂可能是一种相对安全且有效的猕猴成纤维细胞处理方法。核移植前对猕猴成纤维细胞进行处理，有助于优化体细胞核移植技术，也是改善体细胞核在克隆胚中重编程效率的重要途径。 |
| 英文摘要 | The successful cloning of mammals with somatic cell nuclear transfer (SCNT), especially the mice cloned from terminally differentiated lymphocytes and pos-mitotic olfactory neurons, provided unequivocal evidence for the nuclear totipotency of terminally differentiated cells. The SCNT technology has been successfully used in many species cloning, in production of transgenic, gene-knockout animal and model of human disease, and induced the breakthrough in therapeutic cloning and regenerative medicine with stem cells technology, thus holds great prospect for its application in future. However, many urgent problems , such as the low efficiency of success cloning, the developmental abnormalities in cloned embryos and offspring, and the losses throughout gestation, at birth and following birth, should be faced and resolved before the great promise become true. In mammalian preimplantation development, epigenetic reprogramming of DNA methylation, histone modifications and other epigenetic mechanism is essential to the establishment of development totipotency and pluripotency in normal embryo. More investigation on the reprogramming process will help to understand the reprogramming events occurred in cloned embryo; further provide insights into improving the reprogramming and development of cloned embryos. Rhesus monkey, an important kind of lab animal, is very significant for research of disease models and biological medicine. This study mainly focused on epigenetic reprogramming in cloned rhesus monkey embryo and treatment of somatic cells before nuclear transfer. The results were shown as follow: 1), the epigenetic reprogramming events, global methylation dynamics and histone H3 lysine 9 acetylation changes, were firstly described in nonhuman primate. Paternal genome was rapidly demethylated after fertilization and before DNA replication. Maternal genome was just gradually demethylated from 2-cell to early morula stage. Remethyaltion occurred in late morula and resulted into an asymmetric pattern of DNA methylation between hypermethylated trophectoderm and hypomethylated inner cell mass, which is contrary to the asymmetric pattern found in other mammals. 2), most cloned embryos were abnormal or delayed in epigenetic reprogramming. Many 2-cell (667%) and 8-cell cloned embryos showed higher methylation staining than their counterparts in IVF group. The methylation level of ICM nuclei in cloned blastocyst was always higher than IVF control, which might attribute to the development failure of cloned embryos after transfer into surrogate females. 3), In the experiment on treatment of donor cells before nuclear transfer, we found that several methods, including serum starvation and cycle inhibitors (DMSO, roscovitine, aphidicolin and indirubin), were all efficient in synchronizing cell cycle into G0+G1 stage. With BrdU labeling, their inhibitory effect on cell proliferation had been proved, moreover their effect was proved to be reversible. By TUNEL analysis, serum starvation induced more apoptosis in adherent cells (6%), whereas treatment with cycle inhibitors didn’t increase the occurrence of apoptosis compared to cycling cells control, which suggested these inhibitors treatment may be safe and efficient methods for synchronization of rhesus fibroblasts. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6112]  |
| 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 | 杨纪峰. 猕猴体细胞核移植影响因素的研究：着床前胚胎表观遗传重编程和供体细胞周期同步化[D]. 北京. 中国科学院研究生院. 2007. |
入库方式： OAI收割
来源：昆明动物研究所
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