猕猴胚胎干细胞的分离建系和猕猴胚胎干细胞microRNAs表达谱的研究
文献类型：学位论文
| 作者 | 魏强 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-05 |
| 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 孤雌发育 MicroRNAs |
| 其他题名 | Derivation of Rhesus Monkey Embryonic Stem Cells and Characterization of MicroRNAs Signature |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 胚胎干细胞（ESCs）具有自我更新能力和多能性潜能，被认为是研究发育生物学和再生医学理想的体外模型。猕猴(Macaca mulatta)无论在生理上还是病理上都是与人类最为接近的实验动物，也是研究的最为深入的非人灵长类动物，因此猕猴胚胎干细胞（rESCs）是研究ESCs自我更新，定向分化机理等基础理论和细胞替代治疗等临床应用最有价值的研究工具。但是，目前分离建系的rESCs和rESCs的基础研究很少，极大限制了rESCs的应用。本实验中，我们分离建系了三株来自于体外受精囊胚的rESCs和两株来自于孤雌激活囊胚的rESCs，并利用深度测序技术（Solexa sequencing），建立了这两类rESCs的miRNAs表达谱，并对其做了系统比较分析。主要结论如下：（1）rESCs具有与人类ESCs相同的集落形态学特征，rESCs表达灵长类ESCs特异的转录因子和细胞表面标记，具有碱性磷酸酶活性。rESCs在体内体外都具有分化为分别代表外胚层，中胚层和内胚层来源的分化细胞的能力。rESCs在体外经过长期培养，仍然能够保持正常的二倍体核型。说明我们分离建系的rESCs具有自我更新能力和多能性潜能。（2）rESCs高表达诸如miR-302-367 cluster，miR-17 cluster，miR-106a cluster，miR-182/miR-183/miR-96 cluster 和 miR-143/ miR-145 cluster等。rESCs具有与人类ESCs类似的miRNAs表达谱，但是miR-371–373 cluster和C19MC cluster 的表达却与人类ESCs有显著差异。（3）孤雌激活rESCs上调表达let-7 family, miR-374 family, miR-138, miR-195, miR-19a 和 miR-200a等oncogenic miRNAs，而下调表达miR-222和miR-342-3p等tumor suppressor miRNAs。通过分析X染色体相关miRNAs的表达模式发现，两株孤雌激活rESCs中，Pa3具有两条活化的X染色体，而Pa2.2只具有一条活化的X染色体，说明孤雌激活ESCs的两条X染色体虽然都来自成熟卵母细胞的同一条X染色体，但在ESCs分离培养过程中，仍然有可能发生X染色体失活。 |
| 英文摘要 | Embryonic stem cells(ESCs) possessing self-renewal and pluripotent capacity are considered as the powerful tools for studies on regeneration medicine and developmental biology. Rhesus monkey (Macaca mulatta) is one of the most well-studied non-human primate, due to its genetic and physiological similarity to human. Rhesus monkey embryonic stem cells (rESCs) have been widely used as in vitro model for maintaining of pluripotency and differentiation of ESCs. However, the study and application of rESCs are far behind mouse and human ESCs, largely due to the limited rESC lines available and lack of systematic analysis of rESCs. In this study, we isolated three novel rESCs from in vitro fertilization (IVF) blastocysts, two novel parthenogenetic rESCs from parthenogenesis blastocysts and characterized their miRNA signature by Solexa deep sequencing. The results are shown as follows: (1) rESCs shared common morphological property with other primate ESCs, and expressed high level pluripotent markers. rESCs were capable of differentiating into all three embryonic germ layers in vivo and in vitro. Detailed G-banding analysis revealed that rESCs had the normal diploid karyotype. (2) miR-302-367 cluster，miR-17 cluster，miR-106a cluster，miR-182/miR-183/miR-96 cluster and miR-143/miR-145 cluster were rESCs-enriched miRNAs clusters. rESCs miRNA signatures were very similar to human ESCs, however, miR-371–373 cluster only represented a moderate expression level in rESCs. miRNAs from C19MC were lower than minimum threshold. Both of them were enriched in some human ESCs. (3) Parthenogenetic rESCs specially up-regulated some oncogenic miRNAs, including let-7 family, miR-374 family, miR-138, miR-195, miR-19a and miR-200a. Addisionally, some tumor suppressor, including miR-222 and miR-342-3p, were remarkably down-regulated in parthenogenetic rESCs. The X-linked miRNAs analysis suggested that parthenogenetic rESCs Pa3 possessed two active X chromosomes, the other parthenogenetic rESCs Pa2.2 only possessed one active X chromosome, indicating that the X chromosome inactivation (XCI) were also occurred in parthenogenetic ESCs during derivation and culture, regardless of the identical origin of both X chromosome in parthenogenetic ESCs. |
| 语种 | 中文 |
| 公开日期 | 2011-09-19 |
| 源URL | [http://159.226.149.42:8088/handle/353002/6790]  |
| 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 | 魏强. 猕猴胚胎干细胞的分离建系和猕猴胚胎干细胞microRNAs表达谱的研究[D]. 北京. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：昆明动物研究所
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