猕猴胚胎干细胞神经谱系分化过程中mRNA表达谱和miRNA表达谱分析
文献类型：学位论文
| 作者 | 季爽 |
| 学位类别 | 硕士 |
| 答辩日期 | 2013-05 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 郑萍 |
| 关键词 | 猕猴 胚胎干细胞 神经分化 神经干细胞 深度测序 转录组表达谱 miRNA表达谱 |
| 其他题名 | Analysis of whole-genome mRNA and microRNA profilings during the neural differentiation of rhesus monkey embryonic stem cells |
| 学位专业 | 细胞生物学 |
| 中文摘要 | 胚胎干细胞体外神经分化被广泛应用于神经发生过程的研究，同时也为神经退行性疾病的细胞替代性治疗创造可能。尽管体外神经干细胞的来源以及胚胎干细胞向体外神经分化的方法有很多种，但是获得的大多数神经干细胞都缺乏真正的神经干细胞特性，如具有分化的倾向性、无法产生所有神经谱系的细胞类型或者无法响应图式信号。Elkabetz等从人胚胎干细胞分化得到的玫瑰花环神经干细胞被认为是一种真正意义上的神经干细胞，它能够自我更新并分化得到所有区域的神经细胞。尽管有着巨大的应用前景，但该细胞目前无法实现体外长期维持和扩增，在移植实验中的致瘤风险也限制了其临床应用。上述问题的解决需要对玫瑰花环神经干细胞的自我更新和多潜能性维持的分子机制有更深入的了解。与啮齿类动物相比，猕猴在遗传和生理上与人类更接近，并且长期以来一直被认为是人类疾病和临床治疗研究的理想动物模型。结合本实验室的猕猴资源，我们首先建立了稳定可靠的猕猴胚胎干细胞神经分化体系。然后利用建立的分化平台，对猕猴胚胎干细胞神经分化过程中4个标志性阶段细胞样本（胚胎干细胞，第一代玫瑰花环神经干细胞，第六代玫瑰花环神经干细胞及胶质样神经干细胞）进行深度测序，获得了神经分化过程中mRNA表达谱和microRNA表达谱数据。通过对这些数据进行生物信息学分析，得到了以下主要结果：1）寻找到了各个标志性阶段细胞的特异标记基因，并通过比较发现这些标记基因在人类相似的神经分化阶段具有保守性；2）发现Hedgehog信号通路可能在猕猴胚胎干细胞分化为第一代玫瑰花环神经干细胞中起重要作用；3）猕猴胚胎干细胞神经分化过程中很多基因存在不同的可变剪切方式；4）寻找到了不同分化阶段的神经干细胞的高表达miRNAs，它们可能对神经分化过程中特定阶段的维持有重要作用。这些结果为进一步深入研究猕猴胚胎干细胞神经分化及玫瑰花环神经干细胞的自我更新机制和独特的分化潜能调控提供了大量的基础数据。关键词：猕猴；胚胎干细胞；神经分化；神经干细胞；深度测序；转录组表达谱；miRNA表达谱 |
| 英文摘要 | Embryonic stem cell (ESC) in vitro neural differentiation has been widely utilized to study the regulation of neurogenesis. On the other hand, it also provides feasible access to neural stem cells (NSC) or neurons in the cell replacement therapy of neural degenerative diseases. Different protocols have been developed to derive and propagate neural stem cells from ESCs cultured in vitro. However, most of these NSCs are lack of true neural stem cell properties，i.e. they displayed differentiation bias and failed to generate all cell types of neural lineage, or they lost the ability to respond to regional cue .Elkabetz et al. identified an earlier stage of NSC population during the process of human ESC neural differentiation. R-NSC possesses true NSC properties and is considered as stem cells of neural lineage. Albeit potential in the study of neurogenesis as well as in cell replacement therapy, R-NSC is difficult to be maintained and propagated in culture. Moreover, whether R-NSC exhibits high risk of tumorigenesis following transplantation remains elusive. To overcome these obstacles, intensive study of R-NSC is required in order to understand the molecular mechanisms regulating the self-renewal and multipotency of these cells. However, few work was undertaken and little information was obtained regarding the molecular properties of R-NSC in human and even in mice. Rhesus monkey (Macaca mulatta) has more than 90% (92.5% to 95%) DNA homolog to human and has long been considered as a reliable non-human primate model to study various human diseases and to assess the preclinical safety of medical treatments. In the study, we examined by deep sequencing the dynamic transcriptome and miRNAome changes during the differentiation of rhesus embryonic stem cells (rESCs) into the neural lineages. In the study, we examined by deep sequencing the dynamic transcriptome and miRNAome changes during the differentiation of rhesus embryonic stem cells (rESCs) into the neural lineages—neural rosette cells passage 1 (R-NSCP1), R-NSC passage 6 (R-NSCP6), and neural progenitor cells (NPCs). We identified Hedgehog signaling pathway are closely associated with the transition from ESCs to R-NSCP1. Besides, many alternative splicing switches were also found during state transitions. Referring to miRNAomes, many miRNAs were found to be absent in NPCs, leading to the activities of some signaling pathways, such as Wnt signaling pathway. Besides, we also detected miRNAs with high expression variations among lineages, which could be used to maintain specific stages during the neural differentiation.Through the comprehensive bioinformatic analysis of these data, we identified a set of genes as stage-specific markers of neural differentiation, most of which were conserved in human ESC neural differentiation. Many alternative splicing switches were observed during the variable stage transition. We also identified a list of miRNAs with drastic expression level change along with the neural differentiation. The data provided valuable and comprehensive information to further understand the molecular basis regulating the neural differentiation of primate pluripotent stem cells. The candidate genes and microRNAs specifically or highly expressed in R-NSC identified in this study ultimately providing therapeutic solutions for neural degenerative diseases.Keywords: Rhesus monkey; embryonic stem cells; Neural differentiation; Neural stem cells, RNA-seq; Transcriptome; miRNAomes |
| 语种 | 中文 |
| 公开日期 | 2013-05-23 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7434]  |
| 专题 | 昆明动物研究所_哺乳动物胚胎发育 |
| 推荐引用方式 GB/T 7714 | 季爽. 猕猴胚胎干细胞神经谱系分化过程中mRNA表达谱和miRNA表达谱分析[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：昆明动物研究所
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