影响小鼠孤雌胚胎干细胞的建系效率因素初探

目的检测孤雌胚胎干细胞系的建系效率与小鼠品系以及培养体系的关系。方法将小鼠MⅡ期卵子孤雌激活发育至囊胚,然后从囊胚内细胞团分离孤雌胚胎干细胞。结果杂交和近交系小鼠的建系效率没有显著差异,建系的培养体系中加入ERK抑制剂或者采用血清替代品KSR时,建系效率显著提高。结论小鼠孤雌胚胎干细胞的建系效率与小鼠的遗传背景并没有直接关系,而与分离内细胞团的培养体系密切相关。     

家畜胚胎干细胞多能性候选信号通路及分子标志

家畜胚胎干细胞具有重要的生物学意义和广阔的应用前景。以下对比了小鼠、人胚胎干细胞多能性调控信号通路的异同,阐述了小鼠、人胚胎干细胞与家畜胚胎干细胞在多能性分子标志上的差异,并结合本实验室开展绵羊胚胎干细胞研究的实际经验,对目前家畜胚胎干细胞建系中可能存在的多能性候选信号通路及分子标志进行了探讨。     

小鼠类胚胎干细胞分离培养及用于制作嵌合体小鼠的实验研究

目的探讨分离小鼠囊胚内细胞群类胚胎干细胞及用于制作嵌合体小鼠的方法及应用价值。方法分离3.5d小鼠囊胚内细胞群的类胚胎干细胞作为供体细胞,通过显微注射方法将分离的类胚胎干细胞注射到供体小鼠的囊胚腔中,再将注射后的囊胚移植到假孕雌鼠的子宫中制作嵌合体小鼠。结果分离36枚囊胚的内细胞群类胚胎干细胞,注射256只昆明小鼠囊胚中,移植32只假孕雌鼠子宫中,获产崽2窝,共12只,其中2只获毛色嵌合体小鼠。结论采用该技术分离所获得的类胚胎干细胞作为供体细胞制作嵌合体小鼠获得成功,该方法为ES细胞介导的转基因动物制作增添了一条新的途径,在同种不同品系的动物改良及遗传病基因治疗中有一定的应用价值,尤其是对未能建立ES细胞系的大动物的遗传工程操作具有一定意义。     

4个品系小鼠胚胎干细胞系的建立

探索高效的不同品系的小鼠胚胎干细胞的建系方法。B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠,孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG) 人绒毛膜促性腺激素(human chorionic gonadotropin,HCG)促排,3.5天交配后(days post coitus,dpc)冲洗子宫取囊胚,或者2.5dpc冲洗输卵管,卵裂球体外培养获取囊胚。囊胚种植到小鼠成纤维细胞饲养层上干细胞培养液培养,4～5天内细胞团扩增后玻璃毛细管挑出,种植到新的饲养层上过夜再行胰蛋白酶消化,3～4天传代一次。对所建立的小鼠ES细胞系进行形态学、染色体核型、AKP染色、体内外分化能力,干细胞分子标记物荧光免疫染色等鉴定。获得10株小鼠胚胎干细胞,具有典型的胚胎干细胞生长特性,符合ES细胞的鉴定标准。结果表明成功的建立了来自B6D2F1(C57BL/6×DBA/2)、129/SV×DBA/2、C57BL/6、BALB/C等4个不同品系小鼠的10株ES细胞系。内细胞团挑出过夜增殖后消化的培养方法可能有助于提高ES细胞的建系率。     

人胚胎干细胞建系的研究现状与存在的问题

人胚胎干细胞系的建立,对人类胚胎发生和人类发育生物学研究、人类新基因的发现和功能研究以及基因治疗、细胞和组织的移植治疗等领域的突破性进展具有重大意义;回顾了人胚胎干细胞建系研究的历程,就建系的几种方案、路线、意义和可行性进行了探讨;详细系统地说明了迄今为止建立人胚胎干细胞系所需要的饲养层类型、培养基组成、添加细胞因子种类及其作用;分析了建立和维持人胚胎干细胞系所需消化酶的种类及其作用以及目前常用的几种传代方法;从若干方面总结了人胚胎干细胞系的鉴定方法,并对建立和维持人胚胎干细胞系中存在的若干问题进行了剖析,提出了目前急待解决的问题。     

小鼠孤雌胚胎干细胞系的建立及生物学特性鉴定

目的:探讨建立合适的小鼠孤雌胚胎干细胞建系方法。方法:采用氯化锶联合细胞松弛素B激活B6D2F1杂交小鼠卵母细胞,所获得的囊胚与桑椹胚分别用于孤雌胚胎干细胞的建系,观察两者的建系成功率。结果:共建立了12株小鼠孤雌胚胎干细胞系,这些细胞SSEA-1抗原阳性,SSEA-4,TRA-1-81,TRA-1-60表面抗原阴性,具有AKP活性,保持正常染色体核型,体内外分化分别形成畸胎瘤和拟胚体。结论:采用囊胚和去透明带的桑葚胚建立孤雌胚胎干细胞系获得成功。该方法为人类纯合子的胚胎干细胞建系提供基础,在自体细胞治疗领域中具有潜在的应用价值。     

胚胎干细胞起源的探讨

目前胚胎干细胞(ESCs)建系的取材来源包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到类ES样细胞系。这就提出了一个问题,什么是ESCs最接近的体内细胞来源。传统观念常常把ESCs等同于ICM细胞,也有学者认为ESCs更象上胚层细胞,而在已知的分子标记基因方面,ESCs所具有的特征更接近体内早期生殖细胞。不清楚ESCs最接近的体内细胞来源,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一。ESCs系与EG细胞系的分离条件不同表明,加强对ESCs多能性维持基因调控研究具有重要意义。本文从ESCs的经典概念及其发展,早期胚胎细胞和生殖细胞发育规律,早期胚胎细胞、早期生殖细胞和ESCs的关系等方面进行综合分析,认为ESCs可能有多种接近的体内细胞来源。进一步应通过对ESCs建系不同的取材细胞和不同品系的ESCs间进行比较研究,以便弄清ESCs的来源和转化机制,为提高不同物种ESCs建系效率提供理论支持。     

胚胎干细胞建系方法

胚胎干细胞具有的独特特征———分化全能性是生物学各研究领域多年来的热点之一。胚胎干细胞独特的生物学特征在生殖细胞与体细胞及动物个体之间建立了广泛的联系。胚胎干细胞应用前景有着巨大的潜力。为此,对其培养建系的完善性研究显得格外重要。在此,对胚胎干细胞建系方法方面做一简要综述,尤其对建系过程中内细胞团的分离方法及干细胞集落的获取方法进行探讨。     

BALB／c小鼠胚胎干细胞系的建立及其嵌合体小鼠的获得

目的：建立BALB/c小鼠胚胎干细胞系,并用于制作嵌合体小鼠。方法：从BALB／c小鼠囊胚内分离培养内细胞团块。建系后,进行C57BL／6L小鼠受体囊胚腔注射,制作嵌合体小鼠,结果：建立了我国第一株BALB／c小鼠胚胎干细胞系,该细胞系具有典型的ES细胞形态,碱性磷酸酶强阳性,核型正常以及具有分化为三种胚层组织的能力,并已产生5只嵌合体小鼠,结论：建立的BALB／c小鼠胚胎干细胞系具有胚胎干细胞的各种特点,可用于体内外诱导分化研究,在进一步观察生殖系嵌合情况后,决定是否可应用于基因打靶等转基因动物的制作。     

胚胎干细胞起源与本质的探讨

胚胎干细胞(ESC)建系取材包括桑椹胚的卵裂球、囊胚的内细胞团(ICM)、上胚层细胞和原始生殖细胞(PGCs),甚至从新生鼠睾丸细胞也分离得到ES样细胞.传统观念常常把ESC等同于ICM细胞,也有学者认为ESC更像上胚层细胞,而在已知的基因标记方面,ESC所具有的特征更接近体内早期生殖细胞.不清楚ESC最接近的体内细胞类型与本质,可能是制约许多品系小鼠和大多哺乳类动物建系成功率提高的原因之一.综述了胚胎多能性细胞、早期生殖细胞和ESC的研究进展及相互关系,发现ESC可源于多种细胞类型.目前仍难以确定ESC最接近的体内细胞类型,进一步应通过研究不同体内细胞类型源ESC系间的差异,以弄清ESC本质,为提高ESC建系效率提供理论支持.     

Generation of rabbit pluripotent stem cell lines

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate into all somatic cells and tissue lines. They can be genetically manipulated in vitro by knocking genes in or out, and therefore serve as an excellent tool for gene function studies and for the generation of models for some human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, many attempts have been made to generate pluripotent stem cell lines from other species. Comparative characterization of ESCs from different species would help us to understand differences and similarities in the signaling pathways involved in the maintenance of pluripotency and the initiation of differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved across different species. This report gives an overview of research into embryonic and induced pluripotent stem cells in the rabbit, an important nonrodent species with considerable merits as an animal model for specific diseases. A number of putative rabbit ESC and induced pluripotent stem cell lines have been described. All of them expressed stem cell-associated markers and maintained apparent pluripotency during multiple passages in vitro, but none have been convincingly proven to be fully pluripotent in vivo. Moreover, as in other domestic species, the markers currently used to characterize the putative rabbit ESCs are suboptimal because recent studies have revealed that they are not always specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a validated panel of molecular markers specific to pluripotent cells of the developing rabbit embryos. Using rabbit-specific pluripotency genes may improve the efficiency of somatic cell reprogramming for generating induced pluripotent stem cells and thereby overcome some of the challenges limiting the potential of this technology.     

Cloned mice and embryonic stem cell establishment from adult somatic cell

Cloning methods are now well described and becoming routine. Yet the frequency at which cloned offspring are produced remains below 2% irrespective of nucleus donor species or cell type. Especially in the mouse, few laboratories can make clones from adult somatic cells, and most mouse strains never succeed to produce cloned mice. On the other hand, nuclear transfer can be used to generate embryonic stem (ntES) cell lines from a patient's own somatic cells. We have shown that ntES cells can be generated relatively easily from a variety of mouse genotypes and cell types of both sexes, even though it may be more difficult to generate clones directly. Several reports have already demonstrated that ntES cells can be used in regenerative medicine in order to rescue immune deficient or infertile phenotypes. However, it is unclear whether ntES cells are identical to fertilized embryonic stem (ES) cells. In general, ntES cell techniques are expected to be applicable to regenerative medicine, however, these techniques can also be used for the preservation of the genetic resources of mouse strains instead of preserving such resources in embryos, oocytes or spermatozoa. This review seeks to describe the phenotype, application, and possible abnormalities of cloned mice and ntES cell lines.     

哺乳动物体细胞核移植研究进展

体细胞核移植技术已经在基础研究领域与产业化应用领域体现出了重要的价值,因而体细胞核移植技术及其相关研究已经成为了生物领域的持续性研究热点,但是围绕体细胞核移植技术仍然存在许多质疑,其中最主要的就是体细胞核移植的效率较低。尽管如此,体细胞核移植研究仍然在近年来取得了令人瞩目的成就,包括小鼠与恒河猴核移植胚胎干细胞系的建立。该文就体细胞核移植的研究历史与进展进行简要的论述,同时针对体细胞核移植研究中的细胞重编程与治疗性克隆研究中的发展与问题进行剖析,希望能够积极推动治疗性克隆的研究进展,加速核移植与干细胞技术在产业化领域中的应用。     

胚胎干细胞向血液干细胞定向分化的研究进展

骨髓移植是目前治疗恶性白血病以及遗传性血液病最有效的方法之一。但是HLA相匹配的骨髓捐献者严重短缺,骨髓造血干细胞（hematopoietic stem cells,HSCs）体外培养困难,在体外修复患者骨髓造血干细胞技术不成熟,这些都大大限制了骨髓移植在临床上的应用。多能性胚胎干细胞（embryonic stem cells,ESCs）具有自我更新能力,在合适的培养条件下分化形成各种血系细胞,是造血干细胞的另一来源。在过去的二十多年里,血发生的研究是干细胞生物学中最为活跃的领域之一。小鼠及人的胚胎干细胞方面的研究最近取得了重大进展。这篇综述总结了近年来从胚胎干细胞获得造血干细胞的成就,以及在安全和技术上的障碍。胚胎干细胞诱导生成可移植性血干细胞的研究能够使我们更好地了解正常和异常造血发生的机制,同时也为造血干细胞的临床应用提供理论和实验依据。     

The role of stem cells in cardiac regeneration

After myocardial infarction, injured cardiomyocytes are replaced by fibrotic tissue promoting the development of heart failure. Cell transplantation has emerged as a potential therapy and stem cells may be an important and powerful cellular source. Embryonic stem cells can differentiate into true cardiomyocytes, making them in principle an unlimited source of transplantable cells for cardiac repair, although immunological and ethical constraints exist. Somatic stem cells are an attractive option to explore for transplantation as they are autologous, but their differentiation potential is more restricted than embryonic stem cells. Currently, the major sources of somatic cells used for basic research and in clinical trials originate from the bone marrow. The differentiation capacity of different populations of bone marrow-derived stem cells into cardiomyocytes has been studied intensively. The results are rather confusing and difficult to compare, since different isolation and identification methods have been used to determine the cell population studied. To date, only mesenchymal stem cells seem to form cardiomyocytes, and only a small percentage of this population will do so in vitro or in vivo. A newly identified cell population isolated from cardiac tissue, called cardiac progenitor cells, holds great potential for cardiac regeneration. Here we discuss the potential of the different cell populations and their usefulness in stem cell based therapy to repair the damaged heart.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

组蛋白修饰对胚胎干细胞分化过程[]的调控机制

选用人类胚胎干细胞系和由人类胚胎干细胞系分化来的神经干细胞系为研究对象,分析组蛋白修饰对胚胎干细胞分化过程的调控作用。得到了两种细胞系差异表达基因转录起始位点侧翼区域内八种组蛋白修饰的分布模式,以及组蛋白修饰功能簇。研究表明在两类细胞系中,八种组蛋白修饰谱分布模式一致,且呈现两种分布类型; H3K27ac,H3K4me3和H3K9ac组成的功能簇是保守的;H3K27me3,H3K36me3和H3K79me1组成的功能簇以及H3K9me3和H3K27me3组成的功能簇在胚胎干细胞向神经干细胞分化的过程中消失。结果揭示了组蛋白修饰对胚胎干细胞系向神经干细胞系分化过程的部分调控机制,为该分化过程分子调控机制的研究提供部分重要的理论基础。     

Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line

Typically, embryonic stem (ES) cells derived from 129 mouse substrains are used to generate genetically altered mouse models. Resulting chimeric mice were then usually converted to a C57BL/6 background, which takes at least a year, even in the case of speed congenics. In recent years, embryonic stem cells have been derived from various mouse strains. However, 129 ES cells are still widely used partially due to poor germline transmission of ES cells derived from other strains. Availability of highly germline-competent C57BL/6 ES cells would enormously facilitate generation of genetically altered mice in a pure C57BL/6 genetic background by eliminating backcrossing time, and thus significantly reducing associated costs and efforts. Here, we describe establishment of a C57BL/6 ES cell line (LK1) and compare its efficacy to a widely used 129SvJ ES cell line (GSI-1) in generating germline chimeras. In contrast to earlier studies, our data shows that highly germline-competent C57BL/6 ES cell lines can be derived using a simple approach, and thus support broader use of C57BL/6 ES cell lines for genetically engineered mouse models. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

小鼠孤雌胚胎干细胞集落的建立

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