非洲爪蟾（Xenopus laevis）囊胚,神经胚两个cDNA文库的建立

随着分子发育生物学的发展,非洲爪蟾(Xenopus laevis)作为发育生物学,尤其是胚胎诱导、分化机制研究的重要实验材料,受到人们的重视。作者建立的非洲爪蟾囊胚(分期8)和神经胚(分期14)两个不同发育时期胚胎的cDNA文库,为进一步筛选有关的发育基因创造了条件。     

中华大蟾蛛与花背蟾蛛间有性杂交胚胎核型的分析

本文采用胚胎细胞分离及空气干燥法,以痘同发育时期的杂种胚胎为材料,分析了杂种胚胎的核型。结果表明：中华大蟾赊与花背蟾赊间正反组合的杂种胚胎染色体数目均为22条,可配成11对,均由中华大蟾蛛和花背蟾蛛的单倍体组成．枢个组别中,随体显现率,中华大蟾蛛的第6染色体高于花背蟾蛛的第4染色休．从囊胚期到蛾鲜期,杂种胚胎中期相中均有畸变的染色体出现,大多表现为双k4痕及着丝点异位,畸变率为3.8-12%．     

核移植技术研究进展

核移植(nuelear transplation,NT)是将动物早期胚胎或体细胞的细胞核移植到去核的受精卵或成熟卵母细胞中、重新构建新的胚胎,使重构胚发育为与供核细胞基因型相同后代的技术过程,又称动物克隆技术。广义的胚胎克隆技术还包括胚胎分割和卵裂球培养,通常所指的胚胎克隆技术是指狭义概念,即核移植技术。1938年Spmann在所有胚胎细胞都具有与受精卵完全相同、拥有潜在发育全能性的细胞核基础上提出了细胞核移植的概念[‘1。早期核移植实验是在变形虫、蛙、爪蟾、非洲爪蛙等两栖类和鱼类上进行的核质关系研究〔“一“〕,随着胚胎技术的不断进步…     

非洲爪蟾在神经系统疾病研究中的应用

在生物医学研究领域,使用合适的动物模型研究大脑运行机制和疾病机理至关重要。作为经典的脊椎动物模型,非洲爪蟾在研究神经环路构建和功能以及神经疾病的分子机制中具有一定优势。因为非洲爪蟾的胚胎发育过程与人类器官形成过程相似,同时,神经系统疾病往往与神经系统功能异常和发育缺陷有关,所以一些人类疾病在基因和形态上的功能机制可以通过非洲爪蟾的胚胎发育模型进行在体研究,且神经前体细胞的增殖分化以及视觉刺激依赖的神经环路稳态和功能研究已经相对成熟。目前,该模型已经在癫痫和自闭症及表观遗传调控等相关疾病的研究上得到了应用。该文将分别介绍非洲爪蟾蝌蚪模型在环境毒物诱发疾病、神经发育障碍以及表观遗传疾病机制研究领域的最新进展。     

非洲爪蟾眼的形态发生研究

详细观察和描述了非洲爪蟾Xenopus laevis眼的发生和发育变化过程,并分别对各发育时期视网膜的厚度进行了定量分析.非洲爪蟾眼的发牛开始于眼原基的形成,进而形成视泡;晶状体的发生是在视杯外壁增厚的同时诱导覆盖其上的胚胎外胚层内层增厚,形成预定晶状体板;在视网膜和晶状体共同诱导下,预定角膜上皮变为透明的角膜.在视杯出现之前,预定RPE的厚度由厚变薄,NR层不断地增厚直至结构功能完善.     

非洲爪蟾基因MGC64236的克隆及初步分析

MGC64236基因是本实验室用脐静脉内皮细胞免疫的兔血清筛选非洲爪蟾cDNA文库而鉴定的一个功能未知的基因.本研究提取非洲爪蟾受精卵总RNA通过RT-PCR得到基因MGC64236的开放读码框651 bp、编码202个氨基酸;运用生物信息学研究工具进行分析,发现该基因编码的蛋白有3个潜在的跨膜域,有一保守的结构域DUF1370, 可能通过其胞内部分的磷酸化机制在介导细胞内外的信号转导中发挥重要作用;在非洲爪蟾胚胎各个发育时期用RT-PCR检测该基因的表达情况,发现在非洲爪蟾胚胎发育的几个重要时期该基因都有高表达,而在成体则特异地表达于脑和眼等神经组织;构建绿色荧光融合蛋白真核表达载体并转染HEK293细胞, 对MGC64236蛋白的亚细胞定位,发现MGC64236蛋白比较特异地表达在细胞膜.     

花背蟾蜍胎肺发育中表皮生长因子受体的表达和定位作用

取花背蟾蜍(Bufo raddei)363、7、38、39期蝌蚪肺组织和幼蟾肺组织,进行常规石蜡切片,用免疫组化SP两步法检测EGFR的表达。观察了表皮生长因子受体(EGFR)在花背蟾蜍胎肺发育过程的表达特征,并探讨表皮生长因子(EGF)和转化生长因子α(TGF-α)通过与EGFR的作用,对花背蟾蜍胎肺形态发生和肺泡上皮成熟分化的作用。结果表明,36期,EGFR在肺网状隔膜上皮细胞处有表达;37期,肺网状隔膜处EGFR阳性表达很明显,在肺泡囊处表达呈弱阳性;38期,肺网状隔膜处EGFR的阳性表达变弱,在远端的肺泡囊上皮细胞处其阳性表达增强;39期,EGFR在肺泡囊上皮细胞处阳性表达最活跃,在网状隔膜处EGFR的表达很弱;幼蟾期,EGFR阳性反应主要定位在肺泡上皮细胞。结论是,在胎肺发育的不同时期,EGFR在上皮细胞的定位有迁移,免疫组化反应强弱也有差异,说明EGFR在胎肺不同发育阶段发挥不同的功能,它对肺泡上皮细胞的成熟分化有重要调节作用。     

运用反义寡核苷酸研究非洲爪蛙母源基因的功能

母源基因在动物胚胎早期发育中的功能是发育生物学研究领域的难点之一。在过去二十多年里,反义寡核苷酸介导的反向遗传学技术在多种模式生物中均得到广泛应用,在非洲爪蛙母源基因功能分析中,反义寡核苷酸介导的mRNA降解和受体转移(host transfer)技术的联合使用,确立了以转录因子VegT和Wnt信号分子家族成员Wnt11为代表的母源基因在非洲爪蛙胚胎早期图式建成中的重要功能。目前,非洲爪蛙仍然是研究脊椎动物母源基因功能最为方便的模式系统,因此,该文将在简要叙述VegT和Wnt11调控胚胎三胚层的决定与分化和背方组织者中心诱导过程中的作用的基础上,较为详细地介绍反义脱氧寡核苷酸降解母源mRNA的原理和其与受体转移技术结合使用,分析非洲爪蛙母源基因功能的详细技术流程。     

爪蟾(Xenopus laevis)的四倍体及其子一代

在爪蟾受精卵第一次有丝分裂中期进行静液压处理,抑制细胞质分裂从而得到四倍体。实验中获得三只存活了三年以上的四倍体雌性爪蟾。其中一只雌性与二倍体雄性爪蟾作人工催青得到受精卵。现已产卵三批,发育的胚胎400余,经检查子代染色体为三倍体。     

非洲爪蟾GATA-1转录因子与爪蟾发育

GATA－1是GATA结合蛋白（GATA－binding protein）家族的成员之一,正向调节红细胞特异性基因的表达,是红系终末分化所必需的因子。与其他物种不同的是,非洲爪蟾GATA－1转录因子具有两业型,两者结构极为相似,但存在功能上的差异,非洲爪蟾GATA－1转录因子在爪蟾发育过程中起着重要的调节作用。     

非洲爪蟾胚胎细胞核移入花背蟾蜍成熟卵后的细胞学研究

This paper reports the development of the nuclear transplanted eggs obtained by transplanting Xenopus laevis blastomere nuclei to Bufo raddei. The formation of the donor nuclei is similar to that of the male pronuclei in the normal development egg in the early stage while shows degradation in the late stage after transplanting. The chromosome complement formes in some eggs 30 minutes after transplantation, which is assumed to contribute to the development of the transplanted eggs [Acta Zoologica Sinica 51 （5） ： 924 - 931, 2005].     

Cytostatic factor (CSF) in the eggs of Xenopus laevis

Cytostatic factor (CSF) in unfertilized egg cytoplasm causes metaphase arrest when microinjected into zygotes. This was originally described in Rana pipiens eggs In Xenopus laevis, CSF has also been demonstrated. but only when the calcium-chelating agent, EGTA, was injected into the egg cytoplasm. In the present study, however, CSF was demonstrated in Xenopus eggs when donor egg activation was prevented by treatment with CO2 and Mg2+ instead of by EGTA, and recipient blastomere degeneration was prevented by increasing the KCl in the surrounding medium.     

Role of c-myc protein in the early embryonic development of Xenopus

Microinjections of antibodies directed against the protein encoded by the c-myc protooncogene strongly inhibit or arrest the early cell cleavage stage of Xenopus laevis embryos. Injections in one blastomere of a two cell stage embryo inhibit the segmentation of this blastomere. The cleavage of the uninjected blastomere behaves normally. Injections of control rabbit immunoglobulins do not alter the embryonic development.     

Lipid metabolic pathways operating in amphibian full-grown oocytes

The utilization of (2-3H)-glycerol in lipid biosynthesis was analyzed in Bufo arenarum and Xenopus laevis full-grown oocytes. The precursor was more actively incorporated in Xenopus laevis oocytes. Neutral glycerides were the lipids displaying the highest levels of radioactivity followed by phosphatidylcholine and phosphatidylethanolamine. After reincubation of prelabeled oocytes in a saline buffer solution, a net fall in labeled phosphatidic acid concomitant with an increase in phosphatidylcholine were detected. The present findings establish that glycerolipid biosynthesis is operative in full-grown oocytes. In addition, Xenopus laevis oocytes seem to be metabolically more active.     

花背蟾蜍与环境监测

目的分析花背蟾蜍(Bufo Raddei)的分布、生活习性和繁殖行为,并对其进行环境污染物的检测研究进行总结。方法对花背蟾蜍的相关研究文章进行收集,并结合实地调查和观察的数据进行统计分析。结果花背蟾蜍及其蝌蚪在外周血红细胞微核及核异常、血清和肝脏抗氧化酶及脂质过氧化产物MDA、精子结构和活性、蝌蚪生长发育和肾脏结构等方面均表现出对环境污染物的极敏感性。结论花背蟾蜍可作为我国北方环境检测的首选两栖类物种。     

Actin in Xenopus Development: Indirect Immunofluorescence Study of Actin Localization

Actin was studied in Xenopus unfertilized eggs and early developmental stages. Immunochemical proof is given of structural differences between Xenopus laevis muscle actin and nonmuscle cell actin. Actin localization and changes of actin aggregation during Xenopus development were observed using indirect immunofluorescence. We have also tried to explain the presence of an actin shell around the yolk platelets that appeared in our experiments.     

RNA 3' cleavage and polyadenylation in oocytes and unfertilized eggs of Xenopus laevis

Xenopus laevis histone H4 and H1 genes were transcribed in vitro to generate artificial precursor mRNAs (pre-mRNAs). These pre-mRNAs were microinjected into oocytes, matured oocytes, and unfertilized eggs of Xenopus laevis and their 3' cleavage and polyadenylation were investigated. In the oocyte nucleus both H4 and H1 pre-mRNAs were 3' cleaved but were not detectably polyadenylated. In the oocyte cytoplasm there was neither 3' cleavage nor polyadenylation of these histone pre-mRNAs. When injected into either matured oocytes or unfertilized eggs, the pre-mRNAs underwent 3' cleavage but this was inefficient when compared to the oocyte nucleus. In addition approximately 50% of the remaining uncleaved pre-mRNA was subject to a polyadenylation activity which added A tails of approximately 70 A residues. In contrast, artificial mouse beta-globin pre-mRNAs were not detectably 3' cleaved or polyadenylated in either microinjected oocytes or unfertilized eggs.     

Membrane currents in the oocyte of the toad Bufo arenarum

The amphibian oocyte cell model is widely used for heterologous expression of ionic channels and receptors. Little is known, however, about the physiology of oocyte cell models other than Xenopus laevis. In this study, the two-electrode voltage clamp technique was used to assess the most common electrical patterns of oocytes of the South American toad Bufo arenarum. Basal membrane resistance, resting potential, and ionic currents were determined in this cell model. The oocyte transmembrane resistance was 0.35 M(Omega), and the resting potential in normal saline was about -33 mV with a range between -20 mV and -50 mV. This is, to our knowledge, the first attempt to begin an understanding of the ion transport mechanisms of Bufo arenarum oocytes. This cell model may provide a viable alternative to the expression of ion channels, in particular those endogenously observed in Xenopus laevis oocytes.