牛胚胎干细胞建系方法及其影响因素

Establishing bovine embryonic stem cell lines and its influential factors

学术背景:小鼠胚胎干细胞的建系方法已基本成熟,对于建立牛胚胎干细胞无限系的相关报道较少。目的:就牛胚胎干细胞的研究概况、建系方法及影响因素等问题进行概括性论述。检索策略:由该论文的研究人员应用计算机检索Pubmed数据库1980-01/2007-06的相关文献,检索词"bovine,Embryonic Stem Cells,Embryonic Germ Cells",并限定文章语言种类为English。同时计算机检索中国期刊全文数据库1998-01/2007-06的相关文献,检索词"牛,胚胎干细胞,胚胎生殖细胞",并限定文章语言种类为中文。共检索到70篇文献,对资料进行初审,纳入标准:①文章所述内容应与牛胚胎干细胞密切相关。②同一领域选择近期发表或在权威杂志上发表的文章。排除标准:①重复性研究。②Meta分析。文献评价:文献的来源主要是通过对牛胚胎干细胞的研究概况、建系方法及影响因素等方面内容进行汇总分析。所选用的30篇文献中,6篇为综述,其余均为临床或基础实验研究。资料综合:①已有报道从牛鲜胚内细胞团、原始生殖细胞、体外受精胚胎和核移植胚胎中成功分离获得牛类胚胎干细胞。②影响牛胚胎干细胞建系的因素包括胚胎日龄、培养方法、饲养层、细胞因子、传代方法等。③传统的从牛囊胚内细胞团和原始生殖细胞中分离培养胚胎干细胞时会遇到诸多瓶颈,不少学者尝试通过卵母细胞的孤雌生殖和体外成熟、受精来拓宽胚胎干细胞的来源。牛原始生殖细胞的分离过程中,易造成大量原始生殖细胞损失和凋亡,若使用密度梯度离心或特异性荧光抗体标记后通过流式细胞仪进行分离筛选,可能会起到较好的效果。如果使用转白血病抑制因子或干细胞生长因子基因的同源牛胎儿成纤维细胞作饲养层,既可克服外源蛋白污染及动物种间病原交叉感染等问题,同时又提供了培养牛胚胎干细胞所需的外源因子,提高了饲养层的质量。对胚胎干细胞集落进行转基因,通过荧光显微镜可以很清楚地知道所需传代的集落及传代时机。不同学者报道的牛胚胎干细胞碱性磷酸酶的表达有差异,可能与不同的染色程序和细胞分化程度、表型有关,因此碱性磷酸酶活性能否作为牛多能干细胞的特异标记还有待进一步研究。结论:尽管目前有关牛胚胎干细胞的培养与应用技术已取得一定进展,但如何维持牛胚胎干细胞自我复制及多潜能分化的调控机制、如何确定不同的细胞因子联合使用时对牛胚胎干细胞的剂量效应、不同胚胎干细胞系在分化能力上是否存在差异、如何提高利用牛胚胎干细胞制作嵌合体的成功率等诸多问题还有待深入研究。

BACKGROUND： The methods of establishing the mouse embryonic stem cell line have already been basically mature, but the relevance reports about establishing the bovine embryonic stem cell line is relatively less. OBJECTIVE： To discuss the study progress, establishing method and factors of the bovine embryonic stem cells. RETRIEVAL STRATEGY： An online search of PubMed database was undertaken to identify the English articles published from January 1980 to June 2007 using the key words of ＂bovine, Embryonic Stem Cells, Embryonic Germ Cells＂. Meanwhile, the Chinese Journal Full-text Database was searched for the relevant articles published between January 1998 and June 2007 with the same key words in Chinese. Totally 70 articles were collected, and the data were checked in the first trial. Inclusive criteria： （1）Literatures were closely related to bovine embryonic stem cells.（2）Articles announcing in the near future or on authority magazines with the same field were preferred. Exclusive criteria： （1）reduplicative studies.（2）Meta analysis. LITERATURE EVALUATION： The article source is to assemble and analyze the method of establishing the bovine embryonic stem cell line, its influential factors and study progress. Among the selected 30 articles, 6 articles are reviews while others are clinical or basic experimental studies. DATA SYNTHESIS： （1）It has reported that the bovine embryonic stem cells can be obtained successfully from fresh bovine embryo inner cell mass, primordial germ cells, in vitro fertilization embryos and nuclear transplantation embryos.（2）The influenced factors of establishing the bovine embryonic stem cell line include embryo day age, culture method, feeder layer, cell factor, subculture of passage method and so on.（3）There are a lot of bottlenecks in the traditional method of separating and culturing embryonic stem cells from bovine blastocyst inner cell mass and primordial germ cells. Many scholars are attempting to expand the source of embryonic stem cells by oocyte parthenogenesis, in vitro mature and fertilization. It easily leads to a large amount of primordial germ cell loss and apoptosis in the separation process of bovine primordial germ cells. If using density gradient centrifugation or specific fluorescence antibody marker, and screen by flow cytometer, you may get good effect. If using the homologous bovine embryonic fibroblasts that have trans-leukemia inhibitory factor or stem cell growth factor gene as the feeder cells, you may not only overcome exogenous protein contamination and aetiology cross-infection, but also apply the exogenous factor what bovine embryonic stem cells culture need. So the feeder cells can be improved. Transgene to embryonic stem cells mass is observed by fluorescence microscope for defining the mass and the time of subculture. It is possibly relevant with the different dye procedures, cell differentiation degrees and phenotypes that many scholars report the difference about the bovine embryonic stem cells alkaline phosphatase expression. So more studies are demanded on verifying whether alkaline phosphatase activity can be a specific marker of bovine pluripotent stem cells. CONCLUSION： Although the culture and applied technique of bovine embryonic stem cells have already got advanced progress at present, further study are also necessary, such as how to maintain the self-duplication and the regulating mechanism of multipotent differentiation of bovine embryonic stem cells, how to certain the dosages effect to bovine embryonic stem cells by combined use of different cell factors, whether different differentiation abilities exist in different embryonic stem cell lines, and how to improve the success rate that use bovine embryonic stem cells as the chimera.