外胚叶发育不全基因及其受体在斑马鱼牙齿早期发育中的表达

目的 探讨外胚叶发育不全（EDA）基因及其受体（EDAR）基因在斑马鱼早期咽齿发育过程中的表达模式,为进一步研究其在牙齿发育过程中的功能奠定基础。方法 提取受精后48 h（48 hpf）的斑马鱼胚胎总RNA,之后逆转录合成cDNA,以该cDNA为模板,特异性扩增斑马鱼咽齿特异性标记基因dlx2b、Eda信号通路相关基因eda及edar基因片段,用TA克隆试剂盒将这3个基因片段分别连接到pGEMT载体上,将对应质粒用聚合酶链反应（PCR）线性化,以PCR线性化做模板,选择相应的RNA聚合酶体外转录,合成dlx2b、eda及edar基因的反义mRNA探针。收集36、48、56、60、72、84 hpf的斑马鱼胚胎,运用全胚原位杂交技术检测eda及edar基因在斑马鱼咽齿发育部位的表达分布。比较原位杂交结果eda及edar表达区域与斑马鱼咽齿特异性标记基因dlx2b所指示咽齿部位的关系。结果 获得了dlx2b、eda及edar基因的反义mRNA探针。原位杂交结果显示,48~72 hpf时eda及edar在鳃弓牙齿相应部位可见强棕褐色阳性信号,与dlx2b所指示咽齿部位吻合。结论 Eda信号通路配体与受体在斑马鱼咽齿早期发育过程中即牙齿发生至形态发生阶段于咽齿部位特异性强表达,这一结果显示该信号通路可能参与斑马鱼咽齿早期发育的调控。     

Oxytocin receptor signalling modulates novelty recognition but not social preference in zebrafish

Sociality is a complex phenomenon that involves the individual´s motivation to approach their conspecifics, along with social cognitive functions that enable individuals to interact and survive. The nonapeptide oxytocin (OXT) is known to regulate sociality in many species. However, the role of OXT in specific aspects of sociality is still not well understood. In the present study, we investigated the contribution of the OXT receptor (OXTR) signalling in two different aspects of zebrafish social behaviour: social preference, by measuring their motivation to approach a shoal of conspecifics, and social recognition, by measuring their ability to discriminate between a novel and familiar fish, using a mutant zebrafish lacking a functional OXTR. Although oxtr mutant zebrafish displayed normal attraction to a shoal of conspecifics, they exhibited reduced social recognition. We further investigated whether this effect would be social‐domain specific by replacing conspecific fish by objects. Although no differences were observed in object approach, oxtr mutant fish also exhibited impaired object recognition. Our findings suggest that OXTR signalling regulates a more general memory recognition of familiar vs novel entities, not only in social but also in a non‐social domain, in zebrafish.     

Toxicological study of metal and metal oxide nanoparticles in zebrafish

Metal and metal oxide nanoparticles have been widely used in catalytic, electronic and biomedical fields. It is necessary to investigate their toxicity and potential hazards to human and aquatic ecosystems. Zebrafish (Danio rerio), as a promising animal model, has been increasingly utilized to assess the toxicity of nanoparticles. Zebrafish has numerous characteristics for toxicity evaluation, such as short life cycle and high fecundity. This review describes the advantages of using zebrafish in the toxicity assessment of metal and metal oxide nanoparticles. Then we focus on the toxic effects, particularly the acute toxicity and the chronic ones, induced by nanoparticles in zebrafish. Target organ toxicities are also mentioned, including immunotoxicity, developmental toxicity, neurotoxicity, reproductive toxicity, cardiovascular toxicity and hepatotoxicity. The toxic effects of selected metal nanoparticles, including Au, Ag, Cu, and metal oxide nanoparticles such as TiO₂, Al₂O₃, CuO, NiO and ZnO, as well as the underlying mechanisms of nanoparticles causing these effects, are also highlighted and described in detail. Furthermore, we introduce the general factors that affect nanoparticle-induced toxicity in zebrafish. The drawbacks and advantages of using the zebrafish model in nanotoxicity studies are also argued. Finally, we suggest that the application of zebrafish to assess chronic toxicity of metal and metal oxide nanoparticles and the joint toxicity of metal and metal oxide nanoparticles and other pollutants could be hot topics in nanotoxicology.     

基于斑马鱼模型三棱抗血栓活性部位筛选及其化学成分研究

目的：筛选中药三棱抗血栓活性部位并对其化学成分进行研究。方法：采用斑马鱼血栓模型对中药三棱80%乙醇提取物的石油醚部位、乙酸乙酯部位、正丁醇部位和水部位的抗血栓作用进行测试。以硅胶柱色谱等方法对抗血栓作用显著的部位进行分离纯化，通过NMR，MS等方法并结合文献数据鉴定化合物结构。结果：乙酸乙酯萃取物能显著增加斑马鱼血栓模型的心脏染色强度（P<0.01），其余各部位抗血栓作用不明显。在该部位分离得到11个化合物，分别鉴定为对羟基苯甲酸（1）、5-羟甲基糠醛（2）、对甲氧基苯甲醛（3）、对羟基苯甲醛（4）、对香豆酸（5）、阿魏酸（6）、香草酸（7）、原儿茶酸（8）、香兰素（9）、Methyl-3，6-dihydroxy-2-[2-（2-hydroxyphenyl）-ethynyl]benzoate （10）及1，3-O-二阿魏酰基甘油（11）。结论：三棱提取物的乙酸乙酯萃取部位具有显著的抗血栓作用，其主要化学成分为酚酸类化合物。     

非肌性肌球蛋白II-C在斑马鱼胚胎发育过程中的表达*

目的：分析NM II-C在斑马鱼胚胎发育过程中表达的时空变化。方法：收集不同发育阶段的斑马鱼胚胎，应用整体原位杂交技术研究NM II-C在斑马鱼胚胎发育过程中的表达变化情况。结果：受精后17和22小时，NM II-C由原先的广泛表达渐渐集中于前脑、中脑和后脑；受精后26和36小时，NM II-C表达分布到整个中枢神经系统；受精后48小时，NM II-C表达在脊髓变得很弱，主要集中于大脑。结论：揭示了NM II-C基因在斑马鱼胚胎发育过程中的时间和空间上的表达进程，为进一步探讨其在发育中的调控作用提供了实验基础。     

Cellular and molecular mechanisms in the development of a cleft lip and/or cleft palate; insights from zebrafish (Danio rerio)

Human cleft lip and/or palate (CLP) are immediately recognizable congenital abnormalities of the face. Lip and palate develop from facial primordia through the coordinated activities of ectodermal epithelium and neural crest cells (NCCs) derived from ectomesenchyme tissue. Subtle changes in the regulatory mechanisms of NCC or ectodermal epithelial cells can result in CLP. Genetic and environmental contributions or a combination of both play a significant role in the progression of CLP. Model organisms provide us with a wealth of information in understanding the pathophysiology and genetic etiology of this complex disease. Small teleost, zebrafish (Danio rerio) is one of the popular model in craniofacial developmental biology. The short generation time and large number of optically transparent, easily manipulated embryos increase the value of zebrafish to identify novel candidate genes and gene regulatory networks underlying craniofacial development. In addition, it is widely used to identify the mechanisms of environmental teratogens and in therapeutic drug screening. Here, we discuss the value of zebrafish as a model to understand epithelial and NCC induced ectomesenchymal cell activities during early palate morphogenesis and robustness of the zebrafish in modern research on identifying the genetic and environmental etiological factors of CLP.     

斑马鱼皮肤损伤模型的建立及分子机制

目的以皮肤荧光转基因斑马鱼(ktr4:NTR-h Kik GR)为实验动物,研究长春新碱对皮肤细胞的损伤作用及其分子机制,建立皮肤损伤模型。方法取受精后24 h皮肤荧光斑马鱼胚胎,将脱膜后的胚胎分别置于0.01~0.04 mmol·L^-1不同浓度的长春新碱中,24 h后采用荧光显像的方法对斑马鱼皮肤荧光斑点进行计数和统计处理;同时对鱼体细胞死亡情况进行TUNEL试剂盒检测,确定其皮肤细胞死亡的水平;利用蛋白免疫印迹的方法对各组斑马鱼胚胎的caspase-3、KRT4及p53表达水平进行检测。结果在长春新碱与斑马鱼胚胎共同孵育24 h后,0.02、0.04 mmol·L^-1长春新碱明显引起斑马鱼荧光皮肤细胞减少(P<0.01);TUNEL检测表明,长春新碱与对照组比较能明显引起斑马鱼皮肤细胞凋亡;Western bolt分析显示,0.02、0.04 mmol·L^-1长春新碱能诱导斑马鱼胚胎caspase-3和p53表达增加(P<0.01),同时高浓度的长春新碱可使皮肤角蛋白KRT4表达明显减少(P<0.01)。结论利用长春新碱引起斑马鱼皮肤损伤,该局部毒性作用可作为皮肤损伤的造模新方法。     

Nerve growth factor is expressed and stored in central neurons of adult zebrafish

Nerve growth factor (NGF), a member of the neurotrophin family, was initially described as neuronal survival and growth factor, but successively has emerged as an active mediator in many essential functions in the central nervous system of mammals. NGF is synthesized as a precursor pro‐NGF and is cleaved intracellularly into mature NGF. However, recent evidence demonstrates that pro‐NGF is not a simple inactive precursor, but is also secreted outside the cells and can exert multiple roles. Despite the vast literature present in mammals, studies devoted to NGF in the brain of other vertebrate models are scarce. Zebrafish is a teleost fish widely known for developmental genetic studies and is well established as model for translational neuroscience research. Genomic organization of zebrafish and mouse NGF is highly similar, and zebrafish NGF protein has been reported in mature and two‐precursors forms. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the NGF mRNA and protein distribution in the adult zebrafish brain and to characterize the phenotype of NGF‐positive cells. NGF mRNA was visualized by in situ hybridization on whole‐mount brains. NGF protein distribution was assessed on microtomic sections by using an antiserum against NGF, able to recognize pro‐NGF in adult zebrafish brain as demonstrated also in previous studies. To characterize NGF‐positive cells, anti‐NGF was employed on microtomic slides of aromatase B transgenic zebrafish (where radial glial cells appeared fluorescent) and by means of double‐immunolabeling against NGF/proliferative cell nuclear antigen (PCNA; proliferation marker) and NGF/microtube‐associated protein2 (MAP2; neuronal marker). NGF mRNA and protein were widely distributed in the brain of adult zebrafish, and their pattern of distribution of positive perikaryal was overlapping, both in males and females, with few slight differences. Specifically, the immunoreactivity to the protein was observed in fibers over the entire encephalon. MAP2 immunoreactivity was present in the majority of NGF‐positive cells, throughout the zebrafish brain. PCNA and aromatase B cells were not positive to NGF, but they were closely intermingled with NGF cells. In conclusion, our study demonstrated that mature neurons in the zebrafish brain express NGF mRNA and store pro‐NGF.