动物胚胎干细胞的建立与应用

胚胎干细胞(Embryonic stem cells,ES细胞)是从早期胚胎内细胞团(inner cell mass ICM)或原始生殖细胞(Primordial germ cells,PGCs)经体外分化抑制培养分离克隆的。本文介绍了胚胎干细胞的概念,对ES细胞分离、培养和鉴定方法及在动物遗传育种中的应用进行了综述。     

不同因子对花鲈胚胎干细胞增殖的影响

胚胎干细胞(ES细胞)是从动物早期发育胚胎中分离出来的具有发育全能性的一种未分化细胞系。维持花鲈胚胎干细胞(LJES1)的体外生长、增殖及未分化状态需要在培养基中添加一些生长因子。实验通过配制省略1种或多种因子的培养基PESM0～10，计数LJES1细胞在各培养基中增殖的数量，确定各种生长因子对LJES1细胞的增殖作用。同时，对LIF因子和bFGF因子的作用进行了重点的研究，发现LIF因子对早期的LJES1细胞增殖几乎没有作用，其主要作用是维持LJES1细胞的未分化状态，但对晚期LJES1细胞的增殖有一定的作用；bFGF因子对LJES1细胞有强烈的刺激增殖的作用；鲈鱼胚胎抽提液(PEE)以及鲈鱼血清(FS)也促进了LJES1细胞的增殖，2-巯基乙醇(2-ME)具有还原血清中的含硫化合物、防止过氧化物对LJES1细胞的损害及促进贴壁的作用，因而也促进了LJES1细胞的增殖。     

鱼类胚胎干细胞研究进展

胚胎干细胞(Ｅｍｂｒｙｏｎｉｃｓｔｅｍｃｅｌｌ,ＥＳ细胞)是从动物早期发育胚胎内细胞团或原始生殖细胞分离得到的一种未分化的永久性细胞系,它一方面保留了所有的发育潜力,在适合的条件下,能够分化成多种类型的细胞、组织。将ＥＳ细胞移植到动物囊胚后,它可以参与宿主胚胎各种组织的构成,形成嵌合体,直至达到种系嵌合,遗传给后代;另一方面,人们可以对ＥＳ细胞的基因组进行各种遗传操作,包括随机引入外源基因到ＥＳ细胞基因组中,或通过同源重组,定点引入外源基因到ＥＳ细胞基因组中或破坏基因组中某一特定的靶基因;通过细胞移植或核移植技…     

哺乳动物干细胞的研究进展

介绍了干细胞的概念和分类,综述了哺乳动物胚胎干细胞和胚胎生殖细胞的分离培养、生物学特性、定向诱导分化以及几种重要的成体干细胞。讨论了干细胞的应用和存在的问题。     

视黄酸信号通路在青鳉精原干细胞体外增殖与分化中的作用

为探究视黄酸(Retinoic acid, RA)在鱼类精原干细胞(Spermatogonial stem cells, SSC)增殖与分化中的作用,本研究首先以三荧光报告载体pGRY(延伸因子1α启动子驱动组蛋白H2B-绿色荧光融合蛋白、减数分裂联会复合体蛋白3(Scp3)启动子驱动嘌呤霉素红色荧光融合蛋白、精蛋白启动子驱动黄色荧光蛋白)转染青鳉精原干细胞系SG3,获得稳转细胞SG3-pGRY,然后分别在2D与3D培养条件下检测RA信号对其增殖与分化的影响。结果显示,稳转细胞SG3-pGRY的红色荧光可监测细胞内源性Scp3的表达,且细胞仍保持干性及分化潜能,表明其可用于监测细胞分化状态。在2D培养条件下,即在细胞培养板中待细胞生长密度约90%就进行传代培养,RA可显著抑制细胞增殖,其受体α、β、γ泛抑制剂BMS493可促进细胞增殖；第48 h,RA处理可下调细胞多能性相关基因pou5f3、klf4表达,上调减数分裂相关基因dazl表达,但对其他减数分裂相关基因如scp3表达无明显影响；第8 d,处理组及对照组均未能观察到红色荧光,这与已有报道RA处理体外培养小鼠SSC可显著促进Scp3表达,并进入减数分裂I期偶线期的研究结果明显不同。在3D培养条件下,即用96孔球形低吸附微量培养板进行培养,48 h后细胞成球状体聚集生长,RA、 BMS493处理组、对照组在48 h后均观察到明显红色荧光,减数分裂相关基因表达与2D相比显著上调；第8 d与34 d,RA处理组减数分裂相关基因表达均显著高于对照组,而BMS493处理组低于对照组。综上,RA信号可抑制SG3增殖,促进其分化,但不是诱导细胞发生减数分裂的关键分子。本研究不仅为鱼类SSC分化相关研究提供了良好研究模型,而且促进了我们对于RA信号在鱼类SSC增殖与分化中作用的深入认识。     

青鱼的原始器官原基的形成和消化系统呼吸系统的发生

本文介绍了青鱼的早期发育、原始器官原基的形成，以及消化系统、呼吸系统的发生与分化的观察结果。对青鱼胚胎的早期发育和器官发生作了描述；并就此和其他硬骨鱼类的胚胎发育和器官发生进行了比较讨论。     

鱼类原始生殖细胞发育与生殖操作技术研究进展

原始生殖细胞是胚胎发育过程中最早建立的一群生殖干细胞,是有性生殖动物生殖发育的基础。鱼类原始生殖细胞特化遵循“先成论”的模式,早期胚胎发育过程中获得母源生殖质组分的细胞特化为原始生殖细胞,特化形成的原始生殖细胞需要维持其生殖干细胞命运,并经过长距离迁移最终到达性腺原基的位置。原始生殖细胞特化、迁移和命运维持过程受到多种基因和信号通路的综合调控。研究鱼类原始生殖细胞发育不仅有助于深入理解脊椎动物细胞特化、迁移和命运维持等基本生物学过程的调控机理,而且是开发新的养殖鱼类生殖控制和生殖干细胞移植技术的重要基础。本文概述了鱼类原始生殖细胞发育的基础理论以及生殖操作技术研究进展,并展望了未来的发展趋势,以期为开发重要养殖鱼类优良种质创制新技术提供参考。     

中华鳖SOX9基因在胚胎期性腺中的表达模式及在性逆转中的表达变化

SOX9基因在脊椎动物性别决定和性腺分化中扮演重要的调控作用。从mRNA和蛋白水平分析中华鳖SOX9基因在不同组织中的差异性表达、在胚胎性腺和成年睾丸中的细胞定位以及在性逆转中的表达变化,研究SOX9基因在中华鳖性别分化中的调控作用。Real-time PCR结果显示,SOX9基因在中华鳖雄性性腺中特异性表达。免疫荧光染色分析显示,SOX9蛋白在雄性18期胚胎性腺中开始表达,随着性腺的发育,SOX9蛋白定位于性腺Sertoli前体细胞细胞核中;而在雌性胚胎性腺并未见其表达。此外,在雌激素诱导的雄性向雌性性逆转胚胎中,SOX9基因显著下调,而在芳香化酶抑制剂诱导的雌性向雄性性逆转胚胎中,SOX9基因表达则显著上升。研究表明,SOX9基因为中华鳖雄性特异性基因,参与雄性性腺的发育过程,可能在中华鳖早期性别分化过程中起调控作用。     

三种脂质体介导的花鲈胚胎干细胞转化效率的比较

通过3种脂质体(genejammer, genejuice和metafectene)介导绿色荧光蛋白基因转化花鲈胚胎干细胞,探讨不同脂质体介导的花鲈胚胎干细胞（LJES1）的转化效率,以及影响外源基因转化效率的因素,如细胞接种时间、初始接种密度、DNA和脂质体用量以及它们之间的比例等。实验发现,genejammer转化LJES1细胞效率最高,适合于该细胞系的转化。对于同一种脂质体,DNA与脂质体用量的比例对于绿色荧光蛋白基因转化LJES1最为重要,DNA与genejammer的比例为1∶6时,转化效率最高,为27.3%;DNA与genejuice的比例为1∶4时,转化效率最高,为12.1%;而DNA与metafectene的比例为1∶3时,转化效率达到最高,为5.3%。随着DNA、脂质体量的增加,转化效率有所提高,但达到一定的用量后,转化效率反而下降。     

单环刺螠Vasa基因的早期发育表达图式

Vasa基因编码DEAD-box家族成员中一种ATP依赖的RNA解旋酶,是决定生殖系发育的重要调控因子之一。采用同源克隆策略及SMART-RACE技术,克隆了海洋经济螠虫动物单环刺螠Vasa基因的全长cDNA,该序列长4 080 bp,开放阅读框2 322 bp,编码733个氨基酸,具有DEAD-box蛋白家族共有的全部9个保守域。整体原位杂交和半定量RT-PCR结果显示,Vasa基因在未受精卵、受精卵、2～8细胞的早期胚胎中均有明显的表达,显示其母源性提供的特点;从囊胚开始,表达量明显降低,在原肠胚表达信号主要集中在内中胚层细胞中;至担轮幼虫,表达信号进一步集中在消化道处;当发育至体节幼虫时,阳性细胞分布于消化道和体节的隔膜处,进入蠕虫状幼虫,信号仅在头部的腹刚毛附近以及后肠周围的细胞中表达。实验结果为探知刺螠动物生殖系的起源和分化以及低等型生殖腺的发生提供重要的数据。     

坛紫菜雌雄叶状体的细胞分化比较

以室内培养20～90 d的坛紫菜雌雄叶状体为研究材料,用酶解法分别获取单离细胞进行再生培养。在雌雄叶状体的体细胞再生体中,都出现9种不同发育类型。再生体发育类型的数目和比例与种藻日龄密切相关等结果,证实了离体培养的单离细胞发育成不同形态的再生体是基于其离体前处于不同分化时期所致;由壳孢子分化成性母细胞大致可划分成8个不同阶段。雌雄叶状体的细胞分化途经大致相同,但也有一定差异,雌性叶状体的细胞最终分化成雌性性母细胞,并产生大量的丝状体;而雄性叶状体的细胞最终分化成雄性性母细胞,绝大部分生成精子,但极少数产生丝状体。在雌雄叶状体的细胞再生体中,均产生 “类单孢子”并长成正常叶状体。雄性叶状体成熟较雌性早,与其细胞分化速度较快有关。成熟期不同的雌性品系观察结果表明,叶状体成熟越早、生长期越短,其体细胞分化速度也越快。     

Towards obtaining long term embryonic stem cell like cultures from a marine flatfish, Scophtalmus maximus

Culturing pluripotent embryonic stem cells represents a unique model system for in vitro studies of embryo cell growth and differentiation, and represents a connection between in vitro and in vivo manipulation of genes. To further develop and refine stem cell technology for marine fish, we have established cultures of embryonic stem cells isolated from turbot blastulas. The pluripotent nature of our turbot-ES-like cells was supported by their morphology and elevated levels of alkaline phosphatase enzyme activity, their ability to remain undifferentiated for a prolonged culture period, their spontaneous differentiation potential in vitro and their ability to form embryoid bodies (EB) in response to changes in the extracellular environment. In addition, we show that turbot ES like cells express Oct-4 required for the maintenance of pluripotency of ES cells. Cells from 100 blastulas (>10⁵ cells/well) were seeded into gelatine coated 24 well cell culture clusters. The cells were polygonal in shape, with dense cytoplasm and large nuclei. The ES-like cells formed colonies within 24 h following seeding, multilayered in a pyramidal fashion, with maximum cell densities in the middle. The cells proliferated vigorously when seeding densities were high and the cells still had not attached to the gelatine-coated surface. Most of the cells became attached to the surface 48 h following seeding. Attached cells grew more slowly and 20% of the plated colonies could be kept stable for 60 days. Eventually, most of the cultures showed extensive differentiation or died. Only a few cultures (4–5%) survived prolonged culturing (>2 months). The cells were stained for alkaline phosphatase activity, a marker of pluripotency and showed intense staining. More specific, turbot ES like cells in culture expressed Oct-4, detected by immunofluorescence staining. Changing the medium conditions by adding retinoic acid and removing LIF, the proportion of embryoid bodies in our cultures increased. ES-like cells as well as fresh, intact fertilised eggs where successfully cryopreserved. ES cells from the cryopreserved eggs could be isolated and seeded into cultures, forming colonies like the cells from freshly fertilised eggs. Also cryopreserved ES-like cells could be successfully plated. The prolonged survival of these cryopreserved cells has not yet been investigated. The establishment of in vitro cultures of turbot ES-like cells represents a new experimental model for marine flatfish. This revised version was published online in July 2006 with corrections to the Cover Date.     

中华鳖Gper基因克隆、表达及其在精巢中的功能

G蛋白偶联雌激素受体（Gper）是一种膜雌激素受体,介导雌激素的非基因组途径。为研究G蛋白偶联雌激素受体基因（ cDNA全长序列,利用qRT-PCR分析其在不同组织及胚胎不同发育阶段的性腺中的表达模式,并通过来曲唑（letrozole）和Gper抑制剂G-15处理雄鳖初步分析Gper在精巢中的作用。结果显示,中华鳖 cDNA序列全长2023 bp,包含705 bp 5''非编码区、241 bp 3''非编码区和1077 bp开放阅读框,编码358个氨基酸,其氨基酸序列上有7个跨膜结构域和Asp-Arg-Tyr（DRY）三联体结构,基因编码蛋白分子量为41.084 kD,等电点为6.844。表达量变化呈相同趋势：16期表达量最高,随着性腺分化过程表达量显著降低。Letrozole处理组中2表达量明显升高;G-15处理组精巢中,精子发生与促细胞凋亡相关基因表达量显著升高。结果表明,可能参与中华鳖性腺分化早期过程,并调控雄性生殖细胞增殖。     

条斑紫菜叶状体细胞的发育与分化

条斑紫菜壳孢子经室内10～80d培养后长成的叶状体分别被酶解成单个细胞，后者在液体培养基中发育成正常叶状体、畸形叶状体、细胞团、性细胞囊等10种不同类型的再生体，它们的形态与结构、细胞排列与大小，放散单孢子难易程度及最终发育结果等均不同。来自日龄不同的种藻的细胞，其再生体类型的数目和各类再生体的百分数不同。当种藻日龄为10～30d时，在它们的单离细胞再生体中出现了极少的细胞团，正常叶状体和具假根畸形叶状体的百分数随着种藻日龄的增加，急剧减少，但不具假根．的畸形叶状体百分数却随着种藻日龄的增加而增加。当种藻日龄大于40d以后，它们的单离细胞就几乎不能直接再生成正常叶状体。随着种藻日龄的增加，细胞再生体中不具假根的畸形叶状体百分数也显著减少，而细胞团的百分数急剧上升，精子囊和果胞子囊的数量也逐渐上升。来自同一藻体不同部位的细胞，在它们的再生体中，随着细胞所处藻体部位的上移，正常叶状体和畸形叶状体的百分数急剧减少，而细胞团百分数却不断增加。上述结果说明，在离体条件下，条斑紫菜叶状体的单离细胞发育成不同类型的再生体，是由于它们在种藻里处于不同分化阶段的缘故；同时，初步得出在条斑紫菜叶状体的发育过程中，从壳孢子细胞分化成性原细胞的过程可分为8个不同的分化阶段。     

Morphology, endocrinology, and environmental modulation of gonadal sex differentiation in teleost fishes

Successful reproduction by an adult depends on the normal ontogenesis of the gonads, a complex process of cellular and histological differentiation that starts early in life. This process is theoretically predetermined by genetic factors and includes sensitisation of the bipotential gonads to endogenous endocrine factors prior to, during and even after commitment to maleness or femaleness. However, young fish are relatively vulnerable to a host of environmental (physical and chemical) factors that can affect this endogenous endocrine axis, disturbing or even overriding the putative developmental pathway. This sexually lability can be exploited to our advantage for the production of monosex fish populations of the most valuable sex for food production or aquarium fish trade. On the other hand, it represents also a potential path for undesirable influences from endocrine-disrupting chemicals and climatic factors, particularly environmental temperature. This paper provides a detailed account of the early histological process of gonadal sex differentiation, with special reference to gonochoristic species, and reviews the criteria employed to positively identify ovarian and testicular differentiation. It also reviews the development of endocrine competence and sensitivity of the differentiating gonads to exogenous influences in the context of the relative stability of genotypic sex determination in various fish species. Sex differentiation in some species seems to be under strong genetic control and may not require endogenous sex steroid production. Conversely, reliance on endogenous sex steroids for gonadal differentiation is observed in other species and this phenomenon is apparently associated with a higher incidence of environment (mainly temperature)-labile sex differentiation. This revised version was published online in July 2006 with corrections to the Cover Date.     

卵形鲳鲹胚后发育阶段鳃的分化和发育

采用组织学和扫描电镜等方法,鲹研究了卵形鲳(Trachinotus ovatus)胚后发育阶段鳃的分化和发育及其结构和功能的关系。观察发现,仔稚鱼鳃的早期发育可分为3个阶段:第1阶段(0~3日龄)为原基期,鳃原基形成但未分化,鳃耙未出现,仔鱼主要依靠鳍褶、皮肤和卵黄囊上的微血管进行呼吸;第2阶段(4~17日龄)为鳃丝分化、发育期,鳃弓、鳃丝、鳃小片、鳃耙逐渐形成,具备鳃的基本结构和形态特点;第3阶段(18日龄之后)为鳃器官生长发育完善期,鳃弓、鳃丝、鳃小片、鳃耙发育完善,鳃的形态和功能与成鱼相似。进一步研究发现,鳃丝总数随仔稚鱼全长和体质量的增加而增加,单个鳃小片面积和总呼吸面积随仔稚鱼体质量的增加而增大。结果表明,卵形鲳鲳鲹的分化和发育是与仔、稚鱼的生长、形态发育和功能的完善相一致的。     

美洲鲥(Alosa sapidissima)眼睛早期发育的组织学观察

应用石蜡切片及HE染色的方法对美洲鲥(Alosa sapidissima)早期发育过程中眼睛的发生、分化和形成过程进行了系统的观察.结果显示,受精后21 h 13 min,视泡出现;受精后26h 23 min,视泡发育成视杯;受精后35 h 44 min,原始视网膜和晶状体形成;受精后60h 15 min,角膜在视网膜前缘处形成,由单一的立方上皮构成;2日龄仔鱼,巩膜分化完成,由软骨组织和弹力纤维构成,脉络膜出现;3日龄仔鱼,虹膜出现,视网膜分化完全,由外向内的色素层、视觉细胞层、外界膜、外核层、外网膜层、内核层、内网膜层、视神经节细胞层、视神经纤维层和内界膜10层构成,此时视觉细胞层中视杆细胞出现;14日龄仔鱼,角膜分化完全,由自外向内的复层扁平上皮、前弹性层、基质层、后弹性层和内皮层5层构成;22日龄仔鱼,脉络膜腺出现;26日龄稚鱼脉络膜分化完全,由外向内依次为银膜层、血管层和色素层,虹膜也完全分化,由外向内依次为虹膜内皮层、前缘层、基质层、后缘层和色素层;45日龄幼鱼,视网膜内核层分化成两层水平细胞.此时美洲鲥眼睛的各个结构均发育完善.     

Immunocytochemical and ultrastructural identification of adenohypophyseal cells in Ctenopharyngodon idella (Cypriniformes: Cyprinidae) during gonadal differentiation

The adenohypophysis was studied by immunocytochemical and ultrastructural methods in juvenile grass carp (Ctenopharyngodon idella) from natural reproduction in Northern Italian rivers. The adenohypophysis included the rostral pars distalis (RPD), the proximal pars distalis (PPD) and the pars intermedia (PI), all deeply penetrated by branches of the neurohypophysis (Nh). The prolactin (PRL), adrenocorticotropic (ACTH), somatotropic (GH), thyrotropic (TSH), gonadotropic type I (GtH I) and type II (GtH II), somatolactin (SL), melanotropic (MSH) and endorphin (END) cells were identified with antisera raised against piscine and human pituitary hormones. In juveniles of 51–69 mm of total body length (TL) with undifferentiated gonads, the PRL cells, arranged in thick strands, occupied most of the RPD. The ACTH and GH cells organized in cords bordering Nh were, respectively, confined to RPD and PPD. The TSH cells were scattered among ACTH cells in RPD and among GH cells in PPD. Cells simultaneously immunoreactive to anti-follicle stimulating hormone and to anti-croaker gonadotropin were intermingled among GH and TSH cells, which were mostly in the dorsal PPD. The SL cells were detected in PI layers bordering the Nh. The MSH and END cells were intermingled in PI and, unlike what observed in other teleosts, their respective antisera did not cross-react. In individuals of 78–112 mm TL with gonads at the beginning of differentiation, the GtH II cells were detected in PPD; all other cell types increased in number. These results, supported by ultrastructural investigations, suggest that SL and GtH II cells are directly involved in gonadal differentiation in C. idella.