【作者】 岳顺利；

【导师】 刘忠华；

【作者基本信息】 东北农业大学 ， 基础兽医学， 2010， 博士

【摘要】 胚胎干细胞(Embryionic stem cell,ES细胞)在自然状态下并不存在,通常从囊胚内细胞团(ICM)分离得到,与ICM细胞一样ES细胞具有暂时性的自我更新和多种分化潜能的性质。ES细胞是具有分化为外胚层、中胚层和内胚层组织细胞能力的干细胞,是研究细胞“干性”和早期胚胎发育的良好模型,因此广泛地应用于发育生物学、细胞生物学、分子生物学和医学等研究领域。ES细胞被认为是细胞组织移植治疗最有潜能的细胞资源。但是传统分离ES细胞的方法引发了伦理方面的问题。最近报道表明可以从着床前小鼠胚胎单卵裂球成功建立ES细胞系,这一突破性进展在一定程度上解决了自体ES细胞临床应用面临的伦理冲突。因此,人们对采用单卵裂球分离自身ES细胞的方法产生了浓厚的兴趣。但是,该方法尚不成熟仍然存在很多问题,如单卵裂球建立ES细胞的成功率相对很低;姐妹卵裂球是否都具有相同的衍生成ES细胞的能力;它们是否有不同的发育命运等等。因此,对单裂球分离ES细胞系进行更为深入的研究,建立适合的培养体系,提高ES细胞的分离效率,对ES细胞临床应用至关重要。本研究以小鼠为模型,深入研究单个卵裂球建立小鼠ES细胞系,优化了培养体系。研究中首先利用MTT法对丝裂霉素C处理后MEF活力变化进行检测。结果显示,第1,3,5代的MEF,丝裂霉素C(10μg/mL)处理2或3hr,其形态良好,活力可稳定维持是制备饲养层较为理想的条件。在MEF共培养体系下,比较了KSOM和mES两种培养液对ES细胞建立的影响,表明KSOM+mES连续培养体系更有利于ICM的形成,且不改变Cdx2阳性细胞(TE)和Sox-2阳性细胞(ICM)之间比率。MEF共培养下采用KSOM及mES连续培养方案,共建立了71个可传至5代以上的ES样细胞系。其中2CBD获得29个ES样细胞系(72.5%),4CBD和8CBD建系率较低分别为30.0%和11.3%。胚胎的发育阶段越高级从单卵裂球建立ES细胞系的成功率越低。此外,本研究通过对干细胞标志分子表达方式和ICM的形成以及ES细胞分离等方面的研究,证明2-细胞,4-细胞和8-细胞胚胎的姐妹卵裂球并不均一,具有不同的发育能力。由于早期胚胎中的分化决定导致了姐妹卵裂球之间多能性的不平等,很可能是导致胚胎单卵裂球建立ES细胞效率较低的原因。此外,本研究中采用MEF共培养体系,大胆比较LIF添加与否对单卵裂球建立ES细胞系的影响。结果表明不论LIF添加与否,2CBD,4CBD和8CBD都可以形成初级扩展生长。尽管胚胎发育率相似,2CBD-LIF和2CBD-control组分别以83.8%和70.0%的比例建立初级扩展生长。可见,饲养层细胞的存在对ES细胞的建立和维持比培养液中添加LIF更为重要。在MEF共培养体系中LIF的添加与否对于建立ES样细胞克隆不是必要条件,而且ES细胞可在不添加LIF的共培养体系中维持自我更新。有趣的是LIF和MEK1/2抑制剂PD98059协作有助于ES细胞的自我更新,在LIF作用下,激活gp130,同时抑制MEK/ERK通路,对于维持ES细胞的自我更新和分化之间的平衡具有重要作用,只有二者之间保持精确的平衡,ES细胞的自我更新才能保证。研究结果表明,PD98059的靶基因ERK1/2可能参与2-细胞阶段母源基因向胚胎基因的转换(MET)并且激活合子基因组。经ES细胞鉴定,结果表明大部分ES样细胞系保持正常的核型并表达多能性的标志分子,可以在嵌合体小鼠中进行生殖系传递。因此从早期胚胎单个卵裂球建立ES细胞系是可行的。随机抽取早期胚胎的单卵裂球,不经任何特殊处理就具有建立ES细胞的潜能。单卵裂球建立ES细胞的成功率与胚胎发育阶段负相关。更多还原

【Abstract】 Embryonic stem (ES) cells which do not exist in nature, usually can be derived from the inner cell mass (ICM) of the blastocyst, are considered equivalent to ICM cells possessing self-renewal and pluripotency capability. ES cells are the only stem cell type able to possess indefinite self-renewal and to differentiate into cellular derivates of ectodermal, mesodermal and endodermal lineages and are formidable models to investigate fundamental aspects of cell stemness and early embryo development. Therefore they are widely used in the areas of Development Biology, Cell Biology, Molecular Biology and Medical Research and so on..ES cell line has been considered as the most potential cell source of cell tissue therapies. But the traditional method of establishment ES cell line is surrounding by many ethical concerns. Recently success in establishing mouse ES cell lines from a single blastomere of preimplantation embryos has led to a new development in stem cell technology. To some extent this breakthrough demonstrates the possibility of personalized ES cells that would eliminate the ethical concerns surrounding therapeutic cloning. As a result, there was a strong interest in a single blastomere derived ES cells for clinical application of deriving personalized ES cells. However, a relatively low success rate in establishing ES cells from a single blastomere and whether all sister blastomeres are equally competent in deriving ES cells, or if they have distinct fate, these issues indicated that the method itself is immaturation and has many problems need to be discovered. Therefore it is crucial for ES cell clinical application to further investigate a single blastomere deriving ES cell; optimize culture system and enhance success rate in establishing ES cells from a single blastomere.The objective of this study was to optimize the condition of establishing embryonic stem cell from a single blastomere of embryo by using mice as animal models. First the study analyzed cell viability and maintence time of MEF after treatment of mitomycin C by means of MTT method, compared effects of treating time of mitomycin C and cell passages on MEF viabilities after mitomycin C treatment. The results suggest that cell viability sustains more stable after 2 hours and 3 hours of mitomycin C treatment at the concentration of 10μg/mL. Meanwhile, MEF vitalities at the first, the third and the fifth passage were proved to be the suitable condition in preparation for MEF feeder layers.Under the MEF co-culture system we compared the effect of KSOM and mES media on the establishment of ES cells. Our results suggested KSOM is better to enhance mouse single blastomere development, blastocyst development, attachment and the establishment of embryonic outgrowth. We had adopted the co-culture system of mouse ES cells and modified with sequential medium. The continuous co-culture system can promote ICM formation without change the ratio of Cdx2 positive cells (TE) and Sox-2 positive cells (ICM). In total, 71 mES-like cell lines which can be subcultured to up 5 passages have been established in KSOM and mES continuous co-culture system. Among them, there is 29 ES-like cell lines are derived from a single blastomere of two-cell embryos (72.5%). The ES-like cell lines establishment efficiency from 4CBD and 8CBD are much lower than 2CBD (30.0% and 11.3% separately).The more advanced stage embryo resulted in the lower success rate in establishing ES cells from single blastomere. According to expression pattern of stem cell markers, the formation of ICM and the derivation of ES cells, we demonstrated that sister blastomeres of two-, four- and eight-cell embryos were not identical and were not equally competent. Those suggest that the low efficiency in ES cells establishment from a single blastomere of embryos was due to the differentiation in early embryos that resulted in unequal pluripotence competent among sister blastomeres.We also compared the effect on deriveing ES cell line from a single blastomere of embryo with or without LIF. Our results indicated 2CBD, 4CBD and 8CBD can derive primary outgrowth no matter addition of LIF or not. Though the ratio of embryo development of 2CBD-LIF and 2CBD-control is similarity, the ratio of primary outgrowth of them is 83.8% and 70.0% separately. So the addition of LIF is not necessary for establishing ICM-derived ES cell-like colonies in the presence of fibroblast monolayer, and established ES cells can be maintained in an LIF-free medium. Interestingly, blockade of MEK/ERK activity in combination with increased gp130 signalling might promote the self-renewal of ES cell lines. We investigated the effect of inhibition of MEK/ERK signalling (PD98059) combined with increasing stimulation of gp130 signalling by LIF on derivation of ES cells. The result suggested the cooperation of LIF signal and MEK/ERK signalling is essential to the self-renewal of ES cell. Our finding suggested that ERK1/2 which is a target of PD98059 might involve in maternal embryonic transition (MET) and the activation of zygotic genome at the two-cell stage. Most cell lines maintained normal karyotype and expressed markers of pluripotency, including germline transmission in chimeric mice. Our results suggest that the single cells of all early-stage embryos have the potential to be converted into ES cells without any special treatment. The ES cell lines establishment efficiency from a single blastomere of embryo is inversely proportional to the embryo development stages.更多还原