斑马鱼变态反应模型的建立与评价

目的 为系统研究变态反应的相关机制,构建斑马鱼变态反应模型并完善模型评价体系。方法 野生型蓝斑马鱼麻醉后使用微量注射器行腹腔注射,阴性对照组注射鱼用生理盐水,实验组注射肥大细胞致敏剂C48/80化合物,注射3～5min后,无菌解剖取肠道,使用微量毛细管穿刺尾静脉取血。使用行为学、组织病理学、血清学指标定量及半定量评价斑马鱼致敏状态,包括观察并记录斑马鱼注射后行为学改变;取肠道组织标本制备石蜡切片,进行HE染色、PAS染色及甲苯胺蓝染色评价病理改变,并依据评分标准进行半定量分析;使用动力学监测法测定斑马鱼血清中类胰蛋白酶(tryptase)水平。结果实验组斑马鱼在腹腔注射后出现明显的行为学改变,低剂量和高剂量实验组中60.0%～80.0%的斑马鱼出现鱼鳍颤动、身体抽动、剧烈游动等强烈的行为学改变,而对照组中仅有60.0%出现鱼鳍颤动、20.0%出现身体抽动和剧烈游动;实验组肠道组织切片HE染色多见肠道组织病变,PAS染色结果可见杯状细胞聚集分布,实验组肠道病变评分结果显著高于阴性对照组(P<0.05),甲苯胺蓝染色可见肥大细胞数量相较阴性对照组明显升高(P<0.05);实验组...     

HCV非结构蛋白NS5A基因的斑马鱼肝脏表达模型

目的建立HCV非结构蛋白NS5A基因在斑马鱼肝脏特异表达的模型,研究HCVNS5A的体内作用机制并初步探讨HCVNS5A对肝病理标志基因表达的影响。方法克隆肝基因表达调控序列——斑马鱼脂肪酸结合蛋白基因增强子序列（eFABP）和HCV非结构蛋白基因NS5A序列,利用CMV启动子序列,构建HCVNS5A基因和报告基因绿色荧光蛋白基因eGFP在肝脏共表达的基因构件pFC-5AiR。经细胞转染实验验证所建构件的报告基因DsRed表达红色荧光蛋白后,将该基因构件线性化并经显微注射导入斑马鱼胚胎;通过荧光显微镜观察和整体原位杂交技术对目的基因NS5A的表达进行定位,验证其肝脏特异性表达。采用RT-PCR方法和westernblot方法对HCVNS5A基因的转录和翻译水平进行检测;并检测肝脏病理相关的标志基因的转录水平变化。结果细胞转染实验证明基因构件pFC一5AiR的报告基因DsRed能够在肝癌细胞Huh7中表达;斑马鱼胚胎注射该基因构件后在肝脏能够观察到红色荧光蛋白基因DsRed的表达;RT-PCR和westemblot结果显示HCV基因NS5A能够在斑马鱼体内正确表达;原位杂交结果进一步证明了NS5A的表达集中在斑马鱼肝脏内。进一步RT-PCR检测表明HCVNS5A在斑马鱼体内的表达可导致一些肝代谢和纤维化相关基因的上调,如Adiponectin、LDLR、Argsyn、TGF-β和HMGR等,推测NS5A在肝脏脂肪病变和纤维化中具有一定作用。结论成功建立了斑马鱼肝脏表达HCVNS5A的模型;初步数据表明HCVNS5A在斑马鱼体内的表达与部分肝代谢和纤维化标志基因的异常表达相关;该模型可以用于HCVNS5A的体内病理机制的研究。     

斑马鱼模型在肥大细胞研究领域的应用

肥大细胞是重要的区域免疫细胞，参与免疫调节、抗感染和组织损伤修复以及止凝血等病理生理过程，也与过敏、自身免疫性疾病和肿瘤等多种疾病的发生发展密切相关。基础研究受限于肥大细胞仅存在于局部组织，胞内颗粒易溶于水以及标记困难等问题，寻找合适的模式生物成为肥大细胞体内研究的亟需。基于斑马鱼系统固有的发育模式和便于成像的优势，其已广泛应用于生命科学研究领域。该文主要介绍斑马鱼肥大细胞的特征及其作为重要的动物模型工具在肥大细胞生物学和功能研究中的应用价值。     

斑马鱼——髓鞘损伤与再生研究的理想动物模型

脱髓鞘疾病是以多发性硬化为主要表现的自身免疫性疾病,髓鞘脱失与再生的机制不明,利用动物模型进行深入研究是一个有效途径.新型模式动物——斑马鱼具有发育快速、胚胎透明、遗传学技术成熟等优势,通过构建转基因斑马鱼可以对细胞进行活体、动态观察和基因表达模式分析,是研究髓鞘损伤与再生的理想动物模型.     

胚胎期铅暴露对斑马鱼胚胎及幼鱼NMDA受体mRNA表达的影响

目的:了解胚胎期铅暴露对斑马鱼胚胎及幼鱼N-甲基-D-天冬氨酸(NMDA)受体mRNA表达的影响。方法:野生型AB品系斑马鱼胚胎醋酸铅暴露浓度分别为0、0.1、0.5、2.5和12.5 μmol/L,提取各组受精后24、48、72、96和120 h(hpf)斑马鱼胚胎或幼鱼总RNA,实时定量PCR检测NMDA受体亚基NR1.1、NR1.2和NR2B的mRNA表达量。结果:(1)对照组NR1.1和NR1.2及NR2B表达量在胚胎发育过程中逐渐升高,在孵化期(72 hpf)表达量增加明显,在幼鱼早期(96 hpf)时达到高峰(与24 hpf时比较, P<0.01),在120 hpf时仍处于较高水平。(2)随着铅暴露浓度增高,NR1.1表达量增加并有高峰前移的趋势,2.5 μmol/L和12.5 μmol/L铅暴露组NR1.1表达高峰期在72 hpf,并且显著高于对照组(P<0.05);铅暴露组NR1.2和NR2B动态表达也呈类似规律,但NR1.2表达高峰期呈平台化趋势,横跨72 hpf至120 hpf阶段,NR2B表达高峰期出现在72 hpf和120 hpf阶段。(3)NR1.1、NR1.2及NR2B mRNA表达量之间Pearson相关系数值分别为r_NR1.1-1.2 =0.681、r_NR1.1-2B=0.637和r_NR1.2-2B =0.514,均有统计学意义(P<0.01)。结论:在斑马鱼胚胎发育过程NR1.1、NR1.2及NR2B mRNA表达水平逐渐升高,在幼鱼早期达到高峰;胚胎和幼鱼阶段NR1.1、NR1.2及NR2B之间mRNA表达水平存在关联;铅有上调NR1.1、NR1.2和NR2B mRNA表达作用并使表达峰期前移,改变了正常的NMDA受体表达规律。     

斑马鱼:构建神经退行性疾病模型的新型模式生物

动物模型是揭示人类发育机制、疾病机理和和进行药物筛选的重要工具。由于斑马鱼具有胚胎透明、神经系统发育迅速、与人类相关基因和信号通路有高度同源性及可应用于高通量筛选等特点,已经被成功用于建立神经退行性疾病研究模型。本文将介绍斑马鱼作为模式生物近年来在构建几种主要神经退行性疾病模型方面的进展和这些模型在相关药物筛选方面的应用。     

雷公藤多苷促进斑马鱼肝脏损伤和氧化应激的实验研究

目的 研究雷公藤多苷诱导斑马鱼肝脏损伤及氧化应激的作用。方法 随机选取正常3 dpf 野生型AB品系斑马鱼,分为7组:正常对照组、溶剂对照组（0.5% DMSO）、雷公藤多苷300、350、400、450和500 μg/ml浓度处理组,每组30尾斑马鱼。雷公藤多苷处理斑马鱼直至120 hpf（3 dpf~5 dpf）,观察记录每个实验组斑马鱼的死亡情况并计算死亡率,用Origin 8.0统计学软件绘制最佳的“浓度-死亡率”效应曲线并分别计算雷公藤多苷对斑马鱼的MNLC和LC10。另随机选取正常3 dpf野生型AB品系斑马鱼,分为5组:正常对照组、对乙酰氨基酚（8 mM）组、雷公藤多苷片42.5、128和383 μg/ml浓度组,每组60尾斑马鱼。药物处理48 h后,每组随机选取30条斑马鱼置于4%多聚甲醛固定,固定后将斑马鱼转移到70%乙醇中,进行脱水、包埋、切片、HE染色和封片,在显微镜下对染色后的斑马鱼切片进行肝脏病理学分析;在雷公藤多苷处理48 h后,每组随机取30尾斑马鱼收集并处理检测样本,检测过氧化氢酶（CAT）、超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量。结果 雷公藤多苷对斑马鱼的MNLC为383 μg/ml,LC10为417 μg/ml。HE染色结果提示:对乙酰氨基酚对斑马鱼有明显肝毒性,随着雷公藤多苷片浓度增加,斑马鱼肝脏组织结构破坏程度增大。对乙酰氨基酚组、雷公藤多苷片42.5、128和383 μg/ml浓度组的斑马鱼体内CAT、SOD活力以及MDA含量,与正常对照组比较,差异无统计学意义（P＞0.05）。结论 雷公藤多苷可通过诱导氧化应激促进斑马鱼的肝损伤。     

快速眼动疗法的治疗研究述评

本文对眼动脱敏和再加工(又称快速眼动疗法,简称EMDR)的治疗研究进行总结,介绍EM DR的治疗阶段和效果以及与其他疗法进行比较的研究现状,揭示进一步治疗和研究的方向。     

大鼠新型挫伤型脊髓损伤模型的建立及评价

目的:制备新型挫伤型脊髓损伤大鼠模型,通过行为学评分和形态学方法进行评价,为进一步研究脊髓损伤的机制及早期治疗提供依据。方法:SD大鼠随机分为对照组和实验组,对照组(A组)采用改良的Allen法制备急性脊髓挫伤模型,实验组在打击的基础上分别受压3s(B组)、5s(C组)和10s(D组),制备新型挫伤型脊髓损伤模型;各组分别于术后1、3、7、14、21d观察其行为学评分和病理学改变;免疫组织化学方法检测促炎因子高迁移率族蛋白B1(HMGB1)在各组不同时间点的表达变化。结果:实验组各时间点BBB评分和损伤程度与对照组比较均有显著差异(P0.05),其中A组和B组术后损伤程度差别微小;C组后肢运动功能障碍明显,脊髓损伤区域出现典型的病理改变;D组损伤最严重,死亡率高。HMGB1表达以损伤C组为典型代表,术后阳性表达明显增多,第3d达高峰,主要表达部位为神经元胞浆和神经胶质细胞核内。结论:采用改良Allen法打击再受压5s制备的脊髓损伤模型能很好地模拟临床实际,且稳定性高、可复制性好,为一种新型挫伤型脊髓损伤模型。     

Expression of strawberry notch family genes during zebrafish embryogenesis

Our previous study suggested a possible role for Sbno1, a mouse homologue of strawberry notch gene during brain development. In this report, we cloned the zebrafish homologues of sbno, and examined their expression pattern during embryogenesis by whole‐mount in situ hybridization. Zebrafish have three sbno genes: one Sbno1 homologue and two Sbno2 homologues, sbno2a and sbno2b. We observed that the expression of sbno1 and sbno2a was initially ubiquitous and gradually became predominant in the central nervous system as development progressed. The expression of sbno2b was observed in non‐neural tissues in contrast to the other two genes. sbno1 and sbno2a exhibited higher expression in distinct regions within the nervous system of pharyngula‐stage embryos, suggesting possible differing roles for sbno1 and sbno2a during later stages of embryogenesis. Together, the observed gene expression patterns suggest an important role of sbno‐family genes during development of the vertebrate central nervous system. Developmental Dynamics 239:1789–1796, 2010. © 2010 Wiley‐Liss, Inc.     

Expression of zebrafish ROR alpha gene in cerebellar-like structures.

Mouse genetic studies have identified several genes involved in cerebellar development. The mouse mutants staggerer and lurcher are functionally deficient for the retinoid-related orphan receptor alpha (ROR alpha) and glutamate receptor delta2 (Grid2) genes, respectively, and they show similar functional and developmental abnormalities in the cerebellum. Here, we report the cloning and expression pattern of zebrafish ROR alpha orthologues rora1 and rora2, and compare their expression pattern with that of grid2. Expression of rora1 and rora2 is initiated at late gastrula and pharyngula stages, respectively. Both rora1 and rora2 are spatially expressed in the retina and tectum. Expression of rora2 was further observed in the cerebellum, as reported for mammalian ROR alpha. In the adult brain, rora2 and grid2 are coexpressed in brain regions, designated as cerebellar-like structures. These observations suggest an evolutionarily conserved function of ROR alpha orthologues in the vertebrate brain.     

A rapid and efficient method of genotyping zebrafish mutants

In order to facilitate high throughput genotyping of zebrafish, we have developed a novel technique that uses High Resolution Melting Analysis (HRMA) to distinguish wild‐type, heterozygous mutants and homogyzous mutants. This one hour technique removes the need for restriction enzymes and agarose gels. The generated melting curve profiles are sensitive enough to detect non‐specific PCR products. We have been able to reliably genotype three classes of mutations in zebrafish, including point mutants, apc^hu745 (apc^mcr), and p53^zy7 (p53^I166T), a small deletion mutant (bap28^y75) and a retroviral insertion mutant (wdr43^hi821a). This technique can genotype individual zebrafish embryos and adults (by tail‐clip) and is applicable to other model organisms. Developmental Dynamics 238:3168–3174, 2009. © 2009 Wiley‐Liss, Inc.     

G2R Cre reporter transgenic zebrafish

The Cre/loxP site-specific recombination system has been widely used to manipulate DNA in vivo and to study gene function in the mouse by inducible transgenic and conditional gene targeting. To fully use this powerful genetic tool in a relatively new animal model, zebrafish, we generated reporter transgenic lines for easy detection of Cre recombinase activity in vivo. The transgenic fish lines, designated G2R, express two fluorescent protein genes, GFP and RFP, under the control of the ubiquitous promoter of the Xenopus EF1 alpha gene. The G2R animals change their color from green to red (G2R) after Cre-mediated recombination and are useful for development of cell type specific Cre transgenic lines and for cell lineage and fate mapping studies in zebrafish.     

Zebrafish as a model for long QT syndrome: the evidence and the means of manipulating zebrafish gene expression

Congenital long QT syndrome (LQT) is a group of cardiac disorders associated with the dysfunction of cardiac ion channels. It is characterized by prolongation of the QT-interval, episodes of syncope and even sudden death. Individuals may remain asymptomatic for most of their lives while others present with severe symptoms. This heterogeneity in phenotype makes diagnosis difficult with a greater emphasis on more targeted therapy. As a means of understanding the molecular mechanisms underlying LQT syndrome, evaluating the effect of modifier genes on disease severity as well as to test new therapies, the development of model systems remains an important research tool. Mice have predominantly been the animal model of choice for cardiac arrhythmia research, but there have been varying degrees of success in recapitulating the human symptoms; the mouse cardiac action potential (AP) and surface electrocardiograms exhibit major differences from those of the human heart. Against this background, the zebrafish is an emerging vertebrate disease modelling species that offers advantages in analysing LQT syndrome, not least because its cardiac AP much more closely resembles that of the human. This article highlights the use and potential of this species in LQT syndrome modelling, and as a platform for the in vivo assessment of putative disease-causing mutations in LQT genes, and of therapeutic interventions.     

Differential expression patterns and developmental roles of duplicated scinderin‐like genes in zebrafish

Scinderin, the closest homologue of the actin‐severing protein, gelsolin, has two similar paralogs (Scinla and Scinlb) in zebrafish. Scinla is abundant in the adult cornea; Scinlb comprises considerably less corneal protein. Here, we show that scinla is expressed in the nose, lens, brain, cornea and annular ligament of the iridocorneal angle; by contrast, scinlb is expressed in the hatching gland, floor plate, notochord, otic vesicle, brain, pharynx, cartilage, swim bladder and cornea. Activity of scinla and scinlb promoter fragments driving the EGFP reporter gene in transgenic zebrafish resembled scinla or scinlb expression. Previously, we showed that reduction of scinla by injection of antisense morpholino oligonucleotides ventralized embryos; here, specific reduction of scinlb expression led to subtle brain abnormalities associated with increased cell death, decreased shhb expression in the floor plate, and slightly reduced eye distance. Thus, scinla and scinlb have different expression patterns and developmental roles during zebrafish development. Developmental Dynamics 238:2633–2640, 2009. Published 2009 Wiley‐Liss, Inc.     

Emilin genes are duplicated and dynamically expressed during zebrafish embryonic development.

Emilins are a family of extracellular matrix proteins with common structural organization and containing a characteristic N-terminal cysteine-rich domain. The prototype of this family, Emilin-1, is found in human and murine organs in association with elastic fibers, and other emilins were recently isolated in mammals. To gain insight into these proteins in lower vertebrates, we investigated the expression of emilins in the fish Danio rerio. Using sequence similarity tools, we identified eight members of this family in zebrafish. Each emilin gene has two paralogs in zebrafish, showing conserved structure with the human ortholog. In situ hybridization revealed that expression of zebrafish emilin genes is regulated in a spatiotemporal manner during embryonic development, with overlapping and site-specific patterns mostly including mesenchymal structures. Expression of certain emilin genes in peculiar areas, such as the central nervous system or the posterior notochord, suggests that they may play a role in key morphogenetic processes.