| 题名 | 猕猴胚胎干细胞培养以及定向分化成神经细胞的研究 |
| 作者 | 李天晴 |
| 学位类别 | 博士 |
| 答辩日期 | 2005-06 |
| 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 同源饲养层 神经前体细胞 少突细胞 细胞移植 |
| 其他题名 | Culture and Neural Differentiatioii of Rhesus Monkey Embryonic Stem Cells |
| 中文摘要 | 灵长类胚胎干细胞（Es）的研究不仅对理解生殖发育生物学的基础理论、而且对实现细胞替代治疗具有重要意义。当前灵长类ES细胞还有很多问题需要解决，如ES细胞培养体系的改进、ES细胞定向分化成高纯度特定组织细胞的可能性、有效性以及分化机制等问题。本文一方面概述了灵长类ES细胞相关领域的研究进展；另一方面，以称猴胚胎干细胞（rEs）为材料，在改善rES细胞的培养体系、提高其定向分化成神经前体细胞（NPs）和少突细胞的比率进行了研究。实验采用同源称猴细胞系作为饲养层培养rES细胞，并在此基础上利用肝生长因子（HGF）和GS诱导rES细胞定向分化成高纯度的NPs和少突细胞，并评价了NPs是否具有移植功能。主要结论如下：1）四种称猴细胞系（MOF、MESF、MFG和CMESF）可作为饲养层支持rES细胞的生长，保持ES自我更新的能力和分化的多能性。而且这几种称猴饲养层支持ES细胞生长的能力存在明显的差异，进一步的研究表明这些差异主要是由于基因表达种类以及表达量上的差异而导致的。2）采用HGF和GS作为诱导因子添加到NDCM液中，诱导rES细胞分化成可移植的NPs。实验结果如下：单独的HGF或GS仅能诱导ES细胞分化成65±8.3％和69±14％NPs，而HGF和GS的联合使用NPs的比率达到88.3±8.1％，进一步纯化后获得98±1.2％的NPs。获得的NPs能在体内、外分化成三个谱系神经细胞，并能整合到大鼠脑部，发生迁移和分化。25％的NPs细胞8周后仍保持存活状态，其中65-80％的细胞发生了不同程度的迁移，而且细胞在脑部的分化种类以及数目与细胞在体内所处的微环境具有重要的关系。亚克隆实验也进一步证明采用HGF＋GS获得的部分单个NPs具有神经干细胞的特性，也能在体内、外分化成三个谱系的神经细胞，而另外的却只能分化成其中的一、两种谱系的细胞。3）利用五步法，ES细胞能分化成75±6.8％的少突祖细胞和81土8．6％的成熟少突细胞。HGF通过抑制NPs的凋亡和缩短NPs的复制周期，共同促进NPs的增殖。HGF也能诱导HGF+G5获得的NPs分化成少突祖细胞和成熟少突细胞，并且促进少突细胞成熟。而且HGF促进NPs分化成少突细胞的能力受到GS的调控，单独HGF作用将导致ES细胞分化成神经元。本实验的结果为促进rES细胞相关领域的研究，以及ES细胞在神经系统疾病临床应用上提供信息，也为研究NPs和少突细胞的发育提供典型的细胞模型。 |
| 英文摘要 | Study of primate embryonic stem (ES) cells is not only useful for basic research on biomidecal science, but also very helpful for therapeutic cloning. Currently, many questions in the field of primate ES cells are also required for further elucidations, such as the improvement of culture system, highly enriched specific subtypes derived from ES cells and the possible mechanisms of cell differentiation. In order to improve the culture system of rhesus money ES (rES) cells and promote the neural progenitors (NPs) and oligodendrocytes differentiation derived from rES cells, homologous cells as feeders and new neural differentiation system were established in the study. Furthermore, the possibilities of NPs as transplantable donor cells are further evaluated. The main results were as follows. The four homologous feeder cell lines (MESF, MOF, MFG and CMESF) can be used to support the undifferentiated growth and maintenance of pluripotency in rES cells. Comparative studies showed there are different among abilities of the four feeders to support the growth of rES cells, and MOF was preferred homologous feeders, then MESF, CMESF, MFQ respectively. Further studies showed that the abilities variation of the feeders might be related with some genes expression difference. It was found that hepatocyte growth factor (HGF) and G5 as inducers promoted NPs differentiation from rES cells. Alone HGF or G5 only induced rES cells differentiation into 65±8.3% or 69±14% of NPs, respectively. However, the use of HGF and G5 combined markedly increased the NPs differentiation rate (88.3±8.1%). Additional purification resulted in NPs preparations that were 98% nestin positive. In vitro differentiation and in vivo transplantation assays showed that NPs could differentiate into neurons, astrocytes and oUgodendcocytes, The kinds and quantities of differentiated cells derived from NPs were closely correlated with their niches in vivo. Glial differentiation was predominant in periventricular areas, whereas cells migrating into the cortex were mostly neurons. Cell counts showed that 2 months after transplantation, ^25% of transplanted NPs survived and 65-80% of the surviving transplanted cells migrated along the ventricular wall or in a radial fashion. SuMoning demonstrated that several clonal lines derived from NPs expressed nestin and differentiated into three neural lineages in vitro and in rat brains in vivo. In contrast, some subcloned lines showed restricted differentiation both in vitro, and in vivo in rat brains. 3) 75 ±6.8% of oligodendrocyte precursors and 81 ±8.6% of mature oligodendrocytes from rES cells were obtained in five steps using HGF and G5 as our culture system. HGF significantly increased the NPs proliferation speed by both decreasing cell apoptosis rate and shortening cell cycle time in presence of G5. Our studies also revealed that HGF promoted oligodendrocyte maturation by increasing both the length and number of branches and the expression of myelin basic protein. The results from different temporal treatments and combinations of HGF and G5 demonstrated that the ability of HGF responsiveness to initiate oligodendrocyte differentiation was regulated by G5 and alone HGF without G5 induced rES cells differentiation into neurons. Further studies showed that the crucial time point of G5 action was from EBs to NPs.These works are significant for study of the primate stem cells, great helpful for future neural replacement therapies, and provide a platform for probing the molecular mechanisms that control neural induction and oligodendrocyte differentiation. |
| 语种 | 中文 |
| 公开日期 | 2010-10-15 |
| 内容类型 | 学位论文 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6182]  |
| 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 | 李天晴. 猕猴胚胎干细胞培养以及定向分化成神经细胞的研究[D]. 北京. 中国科学院研究生院. 2005. |
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