兔胚胎干细胞分离建系及鉴定以及胚胎干细胞自我更新机制研究
文献类型：学位论文
| 作者 | 王淑芬 |
| 学位类别 | 博士 |
| 答辩日期 | 2007-02 |
| 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 | 兔胚胎干细胞建系 猕猴胚胎干细胞 自我更新 多潜能 信号通路 |
| 其他题名 | Generation and characterization of rabbit embryonic stem cells, and the self-renewal mechanism of embryonic stem cells |
| 学位专业 | 动物学 |
| 中文摘要 | 人胚胎干细胞（ESC）的成功分离培养，吸引大批人对干细胞生物学的关注，特别是ESC在再生医学及人类早期胚胎发育研究的潜在价值。然而在人ESC临床应用之前需要找到合适的动物模型进行大量的预实验研究，从而评价其应用的安全性、有效性及存活效率。因此，从其它物种建立稳定而可用的ESC系也是必不可少的。ESC能无限地自我更新并保持多潜能性，但控制其自我更新的分子机制现在仍然知之甚少且物种间存在差异，了解ESC的自我更新有利于提高建系率、改善培养体系及定向分化体系。本文一方面对ESC分离培养及自我更新机制的研究进展进行了综述，另一方面对以下几个方面的内容进行了研究：1）建立了4株稳定的兔ESC系，能在体外进行长期的培养并保持ESC的多潜能Markers及正常的XY或XX核型，具有碱性磷酸酶活性、表达Oct-4、SSEA-1、SSEA-3、SSEA-4、TRA-1-60及TRA-1-81。与人和小鼠ESC相似，兔ESC表达多潜能基因（Oct-4、Nanog、Sox-2及UTF-1），并表达了与ESC自我更新相关的信号通路（FGF、TGFβ及WNT）的许多基因。从形态来说，兔ESC与灵长类ESC相似，但兔ESC具有较快的增殖能力，与小鼠ESC相类似。在体外及体内兔ESC均能分化成代表原始三胚层的各种细胞类型及组织。2) 从受体抑制实验及生长因子的联合加入可以得出结论FGF及TGF信号通路对维持兔ESC的多潜能性发挥着重要的作用，这样的结果与人ESC相类似。也表明FGF、TGFβ及WNT信号通路在兔ESC的自我更新中都起着作用，而且他们之间可能形成了信号调控网络，相互之间有着正负反馈作用。FGF2+Activin A或TGFβ1+Noggin的无饲养层无血清培养体系不仅能显著抑制兔ESC的分化，且能维持其长期的自我更新。但与小鼠不同，TGFβ信号通路能影响其增殖能力，而对其多潜能性的维持并没有作用。这就更说明了兔比小鼠更适宜成为人类疾病临床治疗之前的模型动物。3）四种猕猴细胞系（MOF、MESF、MFG和CMESF）可作为饲养层比MEFs（小鼠饲养层细胞）更好或同等好支持猕猴ESC的生长，保持其自我更新能力和分化的多能性。而卵泡颗粒上皮样细胞（MFGE）不能支持猕猴ESC的自我更新。进一步的研究表明饲养层支持ESC生长能力的差异可能是由于基因表达种类以及表达量上的差异而导致的。 |
| 英文摘要 | Before clinical applications of embryonic stem cells (ESCs) are begun, extensive preclinical studies must be completed in suitable animal models for assessing safety, efficacy, and long-term survival of transplanted ESC-derived phenotypes. So, establishment of available ESC lines from other animal species will be requisite. ESCs self-renew indefinitely and give rise to derivatives of all three primary germ layers, but the signaling pathways that govern the pluripotency of ES cells remain largely unknown and have species difference. In this thesis, the progress in establishment of ES cell lines, improvements on culture system, and self-renewal mechanism of ESCs have been reviewed, and some research findings have been made in the following: 1) We described the derivation of four stable pluripotent rabbit embryonic stem cell (ESC) lines, one (RF) from in vivo fertilized and in vitro cultured blastocysts and three (RP01, RP02, and RP03) from parthenogenetic blastocysts. These ESC lines have been cultivated for extended periods (RF >1 year, RP01>8 months, RP02>8 months, RP03>6 months) in vitro while maintaining expression of pluripotent embryonic stem cell markers and a normal XY or XX karyotype. The ESCs from all lines expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, SSEA-4), and the tumor-related antigens (TRA-1-60, and TRA-1-81). Similar to human and mouse ESCs, rabbit ESCs expressed pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and signaling pathway genes (FGF, WNT, and TGF pathway). Morphologically rabbit ESCs resembled primate ESCs whereas their proliferation characteristics were more like those seen in mouse ESCs. Rabbit ESCs were induced to differentiate into many cell types in vitro and formed teratomas with derivatives of the three major germ layers in vivo when injected into SCID mice. Our results showed that pluripotent, stable ESC lines could be derived from fertilized and parthenote derived rabbit embryos. 2) Our findings from a combination of growth factor addition and receptor inhibition experiments provide substantial evidence to support the hypothesis that FGF and TGFβ signalling plays key roles in maintaining pluripotency of rabbit ESCs, which is similar to human ESCs. Our findings also show that there is a regulatory network among FGF, WNT, and TGFβ pathway to regulate the pluripotency of rabbit ESCs. Inhibition of FGF and TGFβ pathway result in decrease of rabbit ESCs proliferation rate. Long-term maintenance of rabbit ESCs pluripotency can be achieved with the combination of Activin A or TGFβ1 plus FGF2 and Noggin in the absence of feeder-cell layer and serum. However, in recent report, inhibition of Activin-Nodal-TGFβsignaling by Smad7 or SB-431542 dramatically decreased ESC proliferation without decreasing ESC pluripotency in mouse, which suggests that rabbit ESCs may be a good model for some specific human diseases in preclinical trials. 3) 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, but MFGE can not. In an effort to understand the unique properties of supportive feeder cells, expression levels for a number of candidate genes were examined. These findings showed that the abilities variation of the feeders might be related with some genes expression difference. |
| 语种 | 中文 |
| 公开日期 | 2010-10-14 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6115]  |
| 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 | 王淑芬. 兔胚胎干细胞分离建系及鉴定以及胚胎干细胞自我更新机制研究[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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