猕猴胚胎干细胞自我更新机理及WNT 信号通路在猕猴胚胎早期发育中作用的研究
文献类型：学位论文
| 作者 | 陈栋梁 |
| 学位类别 | 硕士 |
| 答辩日期 | 2009-01 |
| 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 自我更新 胚胎 WNT 信号通路 |
| 其他题名 | Study on self-renewal of rhesus embryonic stem cells and on functions of WNT signaling pathway in the early development of rhesus embryos |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 尽管大部分动物实验是在啮齿类动物上开展的，我们仍然相信涉及人类的许多问题，如胚胎干细胞的体内功能、调亡和肿瘤形成等只有在非人灵长类模型上才能得到最好的回答。猕猴(标准的非人灵长类动物模型)在解剖、生理和代谢方面都和人类非常相似。人类很多神经疾病，如阿尔茨海默氏病、帕金森病，只能在非人灵长类模型上才能精确建模。所以研究猕猴胚胎干细胞自我更新的原理及猕猴胚胎早期发育，对研究免疫排斥，检测基于胚胎干细胞的治疗的可行性，安全性和有效性具有重要意义。本文一方面对胚胎干细胞维持自我更新和多潜能性的机理研究进行了综述，另一方面对以下两个方面的内容进行了研究： 1）运用寡核苷酸芯片和定量PCR验证的方法来分析五株猕猴饲养层细胞的表达模式，期望发现在支持性和非支持性的饲养层细胞中差异性表达的基因。我们着重定位于饲养层胞外空间和细胞膜上的细胞因子，因为这些因子可以通过直接接触或通过膜结合受体激活下游信号通路，并最终促进猕猴胚胎干细胞的自我更新。我们发现在支持性的饲养层中有八个基因是高表达的，他们是GREM2，bFGF，KITLG，DKK3，GREM1，AREG，SERPINF1和LTBP1； 经定量PCR验证的SCF，bFGF和GREM2的表达情况都和芯片数据吻合。 2）为了描述在IVF (in vitro fertilized, 体外受精)，ICSI (intracytoplasmic sperm injection, 单精注射)，SCNT (somatic cell nuclear transfer, 体细胞核移植)和孤雌生殖猕猴囊胚中WNT信号通路的表达情况，我们运用了信号通路特异性PCR Array系统及免疫细胞化学来检测mRNA和蛋白表达水平。其中，ICSI作为IVF胚胎的参照组，以排除显微操作对胚胎质量的影响。结果，我们发现非经典WNT/JNK信号，而不是经典WNT信号通路，在IVF正常胚胎发育中起作用。而体细胞核移植和孤雌生殖的胚胎的WNT信号通路基因表达明显高于正常胚胎。WNT信号通路基因的表达模式可以作为胚胎质量的一个指示标准，有助于回答为什么猕猴SCNT和孤雌生殖胚胎发育异常。 |
| 英文摘要 | While the vast majority of animal research is conducted in rodents, we believe that the unanswered questions regarding ESCs in vivo function, apoptosis, and tumor formation would be best addressed using a non-human primate (NHP). Rhesus macaques (the standard nonhuman primate model organism) possess remarkable anatomical, physiological, and metabolic similarities to humans, and many human neurological diseases, such as Alzheimer and Parkinson diseases, can only be accurately modeled in the nonhuman primate. In this thesis, the mechanism of maintaining self-renewal and pluripotency of embryonic stem cells has been reviewed, and some research findings have been made in the following: 1) We applied the oligonucleotide array and quantitative real-time PCR (qPCR) to analyze the expression profiles of the five rhesus fibroblast cell lines, in the hope of finding out the differentially expressed genes between the supportive and non-supportive feeder cell lines. We investigated the cytokines located in the extracellular region or on the membrane of fibroblast cells, which may interact with rESCs through membrane-binding receptors and trigger the downstream signaling events that finally prolonged the undifferentiated growth of rESCs. Eight genes, GREM2, bFGF, KITLG, DKK3, GREM1, AREG, SERPINF1 and LTBP1, were found to be overexpressed in supportive feeder cells, and three of them, SCF, bFGF and GREM2, were validated by qPCR. 2) In order to depict the WNT expression patterns in IVF, ICSI (intracytoplasmic sperm injection), SCNT and parthenogenetic blastocysts and to figure out whether there are differences between IVF and SCNT blastocysts and between IVF and parthenogenetic blastocysts, we combined the pathway-specific PCR Array System and immunocytochemistry to monitor the WNT expression in both mRNA and protein level. We also tested the WNT signaling pathway components in ICSI bastocysts with the aim of eliminating the effect of micromanipulation. Noncanonical WNT/JNK pathway, instead of canonical WNT pathway, may be active in rhesus IVF blastocysts. Moreover, expression of WNT signaling pathway genes in SCNT and parthenogenetic embryos are significantly higher than those in IVF embryos. |
| 语种 | 中文 |
| 公开日期 | 2010-10-22 |
| 源URL | [http://159.226.149.42:8088/handle/152453/6306]  |
| 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 | 陈栋梁. 猕猴胚胎干细胞自我更新机理及WNT 信号通路在猕猴胚胎早期发育中作用的研究[D]. 北京. 中国科学院研究生院. 2009. |
入库方式： OAI收割
来源：昆明动物研究所
浏览0
下载0
收藏0
其他版本
除非特别说明，本系统中所有内容都受版权保护，并保留所有权利。