牛胚胎生殖细胞的分离与培养

胚胎生殖细胞 (Embryonicgermcells,EG)是由生殖嵴原始生殖细胞 (Primordialgermcells,PGCs)中分离得到的一种未分化而多潜能的干细胞。牛EG细胞的研究在EG细胞核移植、转基因及建立生物反应器方面具有广阔的应用前景。本研究从 2 9- 70日龄牛胎儿PGCs分离得到EG细胞 ,经过抑制分化培养 ,其中一个细胞系传至 6代。所分离得到的EG细胞具有典型的EG细胞形态 ,AP及PAS染色呈阳性 ,核型正常 ,同时观察到这些细胞在体外进行自发性分化 ,可形成类胚体、成纤维样细胞及神经样细胞     

蒙古绵羊原始生殖细胞用于胚胎干细胞培养的研究

原始生殖细胞是用做分离和克隆胚胎干细胞的一种新的细胞资源。本研究将30-45日龄的蒙古绵羊胚胎生殖嵴及其临近组织用机械剪碎和胰蛋白酶+EDTA消化处理,添加DMEM（低糖）+10%FBS（犊牛血清）、38.0℃、5%CO2和饱和湿度条件下进行培养。其结果：未加任何细胞生长因子的情况下与其胎儿成纤维细胞共培养的方式也能分离得到类胚胎干细胞集落。这些集落细胞经多次克隆传代具有胚胎干细胞的诸多特征,如：具有连续传代的能力,细胞集落有典型鸟巢状结构,AKP染色呈阳性,核型分析结果染色体正常等。这些表明该细胞具有多能性,是绵羊类ES细胞。     

胚胎干细胞与胚胎生殖细胞

西北农林科技大学、陕西省干细胞工程技术研究中心的徐小明、杨炜峰、窦忠英等先生对胚胎干细胞(embrgonicstemcells,ES)与胚胎生殖细胞(embryonicgermCells,EG)分别从附置前早期胚胎内细胞团(innercellmass,ICM)和早期胎儿生殖嵴原始生殖细胞(primordialgtrmcells,PGCs)分离克隆出来的一种具有自我更新、无限增殖能力,能分化成代表3个胚层组织细胞能力的干细胞、可对其进行遗传操作、选择和冻存不失其多能性。ES/EG应用于移植医学,在特定条件下可诱导其分化为特定细胞,并可在体外构建特定组织器官,如诱导分化为心肌细胞来治疗心肌梗…     

鸡胚胎生殖细胞在鼠胚成纤维细胞饲养层上的生长

目的:探讨以鼠胚成纤维细胞为饲养层分离、培养鸡胚胎生殖细胞的方法和条件。方法:分离、培养12.5～13.5d鼠胚成纤维细胞。分离孵化5.5d鸡胚原始生殖细胞,原代培养时不使用饲养层,与性腺基质细胞共培养;继代培养时将其置于鼠胚成纤维细胞饲养层上,在含生长因子、分化抑制因子的培养体系中培养胚胎生殖细胞。结果:鼠胚成纤维细胞可连续传代18代以上(4个月),3～15代细胞可以用作饲养层细胞。分离的鸡胚胎生殖细胞在饲养层上可增殖形成典型胚胎生殖细胞集落,并能连续在体外培养超过9代。集落未分化标志高碘酸希夫反应(PAS)呈强阳性,体外分化实验表明胚胎生殖细胞具有多能性。结论:用鼠胚成纤维细胞作为饲养层能获得可连续增殖的胚胎生殖细胞。     

原始生殖细胞体外培养及应用研究

原始生殖细胞是来源于胚胎生殖嵴的一类具有多向分化潜能的干细胞,其形态、细胞表面标志、分化潜能均与来源于囊胚内细胞团的干细胞相似.在饲养细胞层和多种生长因子的共同作用下,可保持原生殖细胞在体外不断增殖而不分化,最终建立EG细胞系.本文就原始生殖细胞体外培养,建立EG细胞系及其应用前景作一综述.     

中国小型猪胚胎生殖细胞的培养和建系

首次报道分别从28天和27天中国小型猪胚胎生殖嵴的原始生殖细胞培养和建立了两株胚胎生殖细胞系(embryonic germ cells,EG cells),各命名为BPEGl和BPEG2。根据它们的生长形态、细胞专一性标志以及体内、外分化等特征,证明了它们具有多能性的特性。这些猪EG细胞具有潜在的应用前景。     

培养原始生殖细胞的新方法

为探讨原始生殖细胞(primordial germ cells,PGCs)在体外长期增殖、生长并长期保持分化潜能的新方法,我们将PGCs分别与睾丸支持细胞(Sertoli cells,SCs)和同源生殖嵴成纤维细胞共培养。结果与SCs共培养的PGCs集落明显多于同源生殖嵴成纤维细胞共培养PGCs集落,传代次数也显著多于同源生殖嵴成纤维细胞．目前与SCs共培养的PGCs已成功传代培养至了第51代。因此我们认为PGCs与SCs共培养,可有效提高原始生殖细胞在体外的增殖能力并可长期维持干细胞的特性。     

人多潜能胚胎生殖细胞的分离和培养

多潜能胚胎性干细胞来源有两条途经,从植入前的早期胚胎内细胞团(inner cell mass,ICM)分离出来的称胚胎干细胞(embryonic stem cells,ES);从原始生殖细胞(primordial germ cells,PGCs)分离得到的称胚胎生殖细胞(embryonic germ cells,EG)。这两种干细胞在小鼠嵌合体实验中,都证明具有参与生殖系传递的能力。这类干细胞在体外保持     

鸡胚胎原始生殖细胞体外培养

以14-15期鸡胚血液为材料,采用Ficoll密度梯度离心方法,提取鸡胚胎原始生殖细胞(primordial germ cells,PGCs),在无基质细胞和基质细胞上分别进行体外培养。从实验结果可以看出：在含有胎牛血清(fetal bovine serum,FBS)、鸡血清(chicken serum,CS)、碱性成纤维细胞生长因子(bFGF)、人胰岛素样生长因子(hIGF-1)、小鼠白血病抑制因子(mLIF)和青,链霉素双抗的M199培养液中培养时,鸡PGCs最多能够存活4天：当采用细胞因子和5天鸡胚胎性腺基质细胞共培养时能存活23代且每代细胞增殖可达近10倍。提纯后的PGCs细胞冻存复苏后,经台盼蓝染色鉴定存活率可达80％左右。     

禽类胚胎生殖细胞的研究

胚胎干细胞有2种来源：一种来自于早期胚胎囊胚期内细胞团（inner cell mass,ICM）的胚胎干细胞（embryonic stem cells,ES细胞）,另一种是来自胚胎生殖腺原始生殖细胞（primordial germ cells,PGCs）的胚胎生殖细胞（embryonic germ cells,EG细胞）。PGCs是生殖母细胞的前体细胞,是精子或卵子的祖先细胞。自Matsui等证实了PGCs同样可以作为胚胎干细胞的原材料之后,现已在人、小鼠、猪和鸡等多种动物的PGCs进行了分离培养并获得了EG细胞。现从形态特征和迁移、分离培养及鉴定方面对禽类EG细胞的研究进展作一综述。     

鸡胚血液中原始生殖细胞的分离及其培养的研究

从孵化４８～５５小时鸡胚中抽取血液,每只胚胎可获血液２～６μｌ左右。一步法离心分离原始生殖细胞,可使其浓度由０．１％以下提高到５０％以上。将多余血细胞用微量吸管移走后,加入添加１０％胎牛血清的ＴＣＭ—１９９做为培养基,３７．５℃,５％ＣＯ２,９５％空气,饱和湿度下培养,原始生殖细胞可成活２４小时左右。     

Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells

During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.     

中华鳖boule基因在生殖细胞中的表达分析

boule基因为DAZ基因家族成员之一,是动物生殖细胞特异表达基因。在哺乳动物中,boule基因的缺失会引起精子生成障碍而导致雄性不育。在无脊椎动物秀丽线虫(Caenorhabditis elegans)中,boule基因同源物的缺失会引起其卵子发生障碍而导致雌性不育。龟鳖动物是最古老的爬行类,是从无羊膜卵到羊膜卵动物飞跃的过渡物种。相比于哺乳类及一些无脊椎动物,目前关于龟鳖动物生殖细胞发育模式的研究还非常有限。因此,本文以中华鳖(Pelodiscus sinensis)为研究对象,以期揭示boule基因对龟鳖动物生殖细胞发育分化的调控作用。首先,利用特异引物克隆获得中华鳖boule基因的cDNA序列,共1 005 bp,其中,3′端非编码区57 bp,开放阅读框948 bp,共编码315个氨基酸。氨基酸序列多重比对分析显示,中华鳖与绿海龟(Chelonia mydas)同源性最高,达92%,与小鼠(Mus musculus)的同源性达83%,与果蝇(Drosophila melanogaster)的同源性达53%,与青鳉(Oryzias latipes)的同源性达42%。反转录实时定量PCR(RT-qPCR)分析结果显示,中华鳖boulem RNA主要在性腺组织精巢和卵巢中表达,而在其他体细胞组织中几乎检测不到表达。原位杂交结果显示,中华鳖boule m RNA在两性生殖细胞中特异表达,且在不同分化时期的生殖细胞中呈动态表达。在精巢中,boule m RNA在初级精母细胞中表达最强,在精原细胞和次级精母细胞中表达较弱,在精子细胞和精子中难以检测到表达信号;在卵巢中,boule mRNA在初级卵母细胞中表达信号最强且信号在初级卵母细胞胞质中均匀分布,生殖细胞发育进入卵母细胞生长期后,信号开始聚集在核周胞质,随着卵母细胞的成熟,信号逐渐变弱。本研究结果表明,boule基因可能在中华鳖两性生殖细胞的减数分裂过程中均具有重要的调控作用。     

高氟对3月龄公鸡生殖细胞的损伤作用

为了探讨高氟对鸡生殖细胞损伤作用,选用3月龄蛋公鸡10只,实验组和对照组各一组,每组5只,实验组在饮水中添加3.25 g/L的氟化钠,对照组饮用自来水,实验周期为120 d。分别于60 d、80 d、100d、120 d采集精液,检测精子密度、早期精细胞微核率、精子畸形率及精子活率。120 d后,处死动物,取两侧睾丸,称重并测量大小。结果表明,氟化钠可致精子数量减少,活率降低,精子畸形率和微核率上升,并呈明显的时间-效应关系。比较两组睾丸重量和大小,差异极显著(P<0.01)。说明高氟对鸡生殖细胞有遗传损伤作用,高氟可能是致突变物。     

Germ cell kinetics in the neonatal rabbit testis

Summary Germ cells in the developing rabbit testis were found to undergo several distinct changes in the first two weeks after birth. Mitotic activity, which had been high in the late fetal period, reached a peak on the day before birth, then diminished steadily and ceased entirely after five days of age. Extensive germ cell degeneration occurred in the first week after birth resulting in accumulation of pools of degenerating germ cells in the central portions of the seminiferous cords. Following shortly after the peak of mitotic activity, germ cells at various stages of preleptotene could be found in squash preparations. This corresponded to the time when germ cells in the rabbit ovary enter and proceed through meiotic prophase. There was no evidence of entry into leptotene or later stages of meiosis in the neonatal testis. The findings suggest that a similar stimulus for entry into meiosis may exist in both sexes, but a blockage occurs in the male.Technical assistance was provided by Margaret Randolph and David Knibbs     

睾丸生殖细胞的凋亡及其调控

睾丸生殖细胞在分化过程中存在自发性和诱发性凋亡,这是清除过量或异常生殖细胞的一种重要途径。生殖细胞的凋亡涉及内分泌、细胞社会组成和基因等多因素的调控。深入了解生殖细胞凋亡的调控机制,明确决定睾丸生殖细胞（Ｇｅｒｍ ｃｅｌｌｓ,Ｇｃ）凋亡机制的分子组成,将为治疗男性不育和开发男性避孕药物奠定理论基础。     

Germ cells in the crustacean Parhyale hawaiensis depend on Vasa protein for their maintenance but not for their formation

Germ cells are a population of cells that do not differentiate to form somatic tissue but form the egg and sperm that ensure the reproduction of the organism. To understand how germ cells form, holds a key for identifying what sets them apart from all other cells of the organism. There are large differences between embryos regarding where and when germ cells form but the expression of Vasa protein is a common trait of germ cells. We studied the role of vasa during germ cell formation in the crustacean Parhyale hawaiensis. In a striking difference to the posterior specification of the group of germ cells in the arthropod model Drosophila, all germ cells in Parhyale originate from a single germ line progenitor cell of the 8-cell stage. We found vasa RNA ubiquitously distributed from 1-cell to 16-cell stage in Parhyale and localized to the germ cells from 32-cell stage onwards. Localization of vasa RNA to the germ cells is controlled by its 3′UTR and this could be mimicked by fluorescently labeled 3′UTR RNA. Vasa protein was first detectable at the 100-cell stage. MO-mediated inhibition of vasa translation caused germ cells to die after gastrulation. This means that in Parhyale Vasa protein is not required for the initial generation of the clone of germ cells but is required for their subsequent proliferation and maintenance. It also means that the role of vasa changed substantially during an evolutionary switch in the crustaceans by Parhyale from the specification of a group of germ cells to that of a single germ line progenitor. This is the first functional study of vasa in an arthropod beyond Drosophila.