斑马鱼二倍及单倍体胚胎干细胞培养和鉴定体系的研究

Owing to their unique features, embryonic stem (ES) cells derived from early developing embryos provide a promising tool for experimental analyses of basic developmental processes such as pluripotency control and cell fate decision. As a model organism, zebrafish are widely used in basic studies in developmental biology and medicine. However, many effective ES cells-based methods and techniques currently used in other model organisms can not be applied in zebrafish duo to the lack of an excellent zebrafish ES cell culture system. Previous attempts of derivation of zebrafish ES cell long-term cultures were not successful. We consider that the failure was because of the less appropriate culture conditions which led to variations in chromosome number and loss of cell pluripotency. Our preliminary data suggest that it is possible to develop zebrafish long-term ES cell cultures in an appropriate culture system optimized by defining the components of medium. In this project, we propose to further improve the culture conditions and derive zebrafish ES cell cultures capable of germ line contribution. The features of cultivated ES cells are to be characterized by cytogenetics analysis, clonal growth, gene transfer experiment, ES cell marker expression analysis, embryoid body formation, intra- and inter- species chimera formation whereby putative stem cells are transplanted into early embryos and their ability to generate different cell types is analyzed in host embryos that provide a proper developmental environment. The successful generation of zebrafish ES cell lines will provide a useful tool for researches such as gene modification based on ES cells. Compared to diploid ES cells, haploid ES cells, which combine haploidy and pluripotency, enable direct genetic analyses of recessive phenotypes in cells because every gene has only one copy in the haploid genome. Haploid ES cells have been elusive for culture due to their inferior growth and possible genomic instability. We have previously generated the medakafish haploid ES cells which gave rise to the production of the first fertile semi-cloned animal. Recently, the haploid ES cell cultures were obtained in mouse. In this project, we also propose to generate the zebrafish ES cell cultures from gynogenetic zebrafish embryos. To characterize the haploid cultures, we will employ the procedure and methods used in medaka hiploid ES cell, such as ES cell marker expression analysis, in vitro differentiation by embryoid formation and in vivo differentiation by chimera formation. For characterizing the haploidy and genetic integrity, we will perform chromosomal binding, flow cytometry analysis, and semi-cloning experiments. Zebrafish haploid ES cell cultures may offer a proof of the ability of zebrafish haploid genome in supporting ES cell growth and developmental pluripotency, and more importantly, may offer an yeast-like system for analyzing recessive phenotypes in numerous cell lineages of zebrafish in vitro.

胚胎干细胞（ES细胞）是生命科学研究的重要素材。然而，斑马鱼作为重要的模式脊椎动物，其ES细胞培养尚未成功，使以ES细胞为基础的研究工作难以开展。我们的前期研究表明，斑马鱼ES细胞培养的主要障碍在于培养条件不完善。本研究拟在前期工作的基础上，优化培养条件，建立有种系传递能力的斑马鱼ES细胞系，探索它们的生长特性、体内外分化潜能及有效的基因转移途径。斑马鱼ES细胞的培养成功将为基因修饰等研究提供有用的细胞平台。本研究还将在二倍体ES细胞研究的基础上，分离和培养雌核发育单倍体胚胎的囊胚细胞，建立斑马鱼单倍体ES细胞培养和鉴定体系。相比于二倍体ES细胞，单倍体ES细胞更适于遗传筛选和基因功能分析等研究，因为其基因组中每一基因只有一份，基因的改变可直接从表型上反映出来。该体系的建立，不仅可在理论上阐明斑马鱼单倍体基因组对ES细胞在体外生长发育的支持能力，而且还将提供一个更有效的斑马鱼基因研究工具。

胚胎干细胞是生物学和医学基础及应用研究的重要材料。斑马鱼胚胎细胞在体外培养中存在缺乏长期生长能力、染色体变异、干性丧失等问题。本项目针对这些问题开展了以下研究，取得了一定的成果。1. 斑马鱼二倍胚胎细胞培养和鉴定体系研究。通过优化培养条件建立了比较完善的斑马鱼细胞培养系统，得到胚胎细胞系ZFESC2。细胞生长稳定，染色体正常，表达干细胞标记。细胞能参与嵌合体生殖腺等组织的发育，在嵌合体内发生分化。本研究基本解决了斑马鱼胚胎培养细胞生长稳定性、基因组稳定性和干性维持等问题，发现klf4和sox2是斑马鱼合适的干细胞标记。2. 斑马鱼单倍胚胎细胞培养鉴定体系研究。斑马鱼单倍体细胞培养更为困难。通过优化了二倍细胞培养系统，得到单倍体细胞系ZFHESC1。细胞基因组稳定，表达标记基因klf4与sox2，具有单克隆生长能力，能在胚状体和嵌合体中发生分化。因此，斑马鱼单倍体基因组能支持干细胞的体外生长和分化。3.鱼类生殖细胞培养及生殖细胞发育机制研究。以斑马鱼胚胎培养体系为基础尝试培养斑马鱼原始生殖细胞(PGC)，发现斑马鱼PGC不能持续扩增。为此，研究中以青鳉为模式探索了鱼类PGC培养条件。在优化培养系统中，青鳉囊胚细胞在体外形成的PGC能持续扩增，传代培养后仍有较强生长能力，培养的PGC能迁移到嵌合体胚胎性腺。但该系统仍不足以支持斑马鱼PGC体外生长，表明斑马鱼和青鳉PGC生长发育条件可能存在较大差异。另外还对microRNA在斑马鱼PGC发育中功能和作用机制进行了研究，发现在生殖细胞发育中有重要作用的microRNA。4. 应用斑马鱼及其它鱼类细胞研究鱼类抗病毒致机制。用斑马鱼和其它鱼类多种细胞系开展了鱼类细胞抗病毒和鱼类病原免疫学的相关研究。建立了抗鱼类病毒药物的高通量细胞筛选平台，从大量的化合物中成功筛选到对鱼类病毒具有抗性的化合物。另外，还进行了鱼类神经坏死病毒致病机制研究，发现海鲈识别病毒感染的RLRs信号通路。本项目研究结果为以鱼类细胞为材料的基础和应用研究提供了重要的工作基础。