斑马鱼综述写作9
斑马鱼(Danio rerio)的资源管理：综述
斑马鱼（Danio rerio）的资源管理：综述Christian Lawrence摘要斑马鱼最近成为了一个卓越的生物医学研究的模型的脊椎动物。
它作为人类疾病和发展的模型，这个同样令人喜欢的特征为它受欢迎做出了贡献；即繁殖力高，体积小，迅速的繁衍周期，在早期胚胎发育早期的光学透明性，也有许多其他学科的研究者长期致力于它的研究，包括动物行为，鱼类生理，水产毒理学。
尽管如此，严谨的饲养斑马鱼的技术还是不够发达。
虽然斑马鱼有一个相当大的身体，都和畜牧业有直接或间接的关系，这个信息有许多不同的来源，而且它很少被应用到发展中国家的标准协议。
这项综述是尝试把可利用的与斑马鱼生物学和文化相关的科学资料整合到一个这项领域的概述，可以用于研究中的这个重要的动物模型的使用效率的改善。
这个综述还强调了在那些领域需要做进一步的研究。
目录1.简介2.斑马鱼的自然历史2.1 喜好的栖息地及分布2.2 繁殖和行为2.3 寿命2.4 食性3.斑马鱼文化3.1 水化学3.1.1 温度3.1.2 PH值3.1.3 硬度3.1.4 盐度3.1.5 溶氧3.1.6 含氮废物4.营养，食性和饲养方法4.1 营养需求4.2 食性4.3 饲养5.繁殖和养殖技术5.1 繁殖5.2 养殖技术5.3 产卵率6.幼体培育6.1 幼体生物学6.2 食性和营养6.3 水质6.4 生长率和存活率7.成体培育7.1 保持密度7.2 遗传育种计划8.总结致谢参考文献1.简介在过去的二十年里，斑马鱼已成为一个研究遗传学和发展的重要的脊椎动物模型，近来，也包括人类疾病和筛查治疗药物。
大量的有利的性质，包括它体积小，快速的发展和繁殖速度，早期发展过程中的光学透明性，比较容易的遗传选育和与人类相似的遗传特点，而且它将很有可能在其他领域的研究中刺激经济的增长，特别是它的基因组草案的进一步完善和令人振奋的用来做扩展研究的可利用的工具和方法。
鉴于斑马鱼作为一个相当重要的实验模型，伴随着的是它大量使用和相关培育设施的建立和维修的大额的经济耗费，在一定程度令人惊讶的是它的畜牧业不发达。
斑马鱼是荧光探针用于生物成像的理想模型
斑马鱼是荧光探针用于分子成像的理想脊椎动物模型Sung-Kyun Ko,a Xiaoqiang Chen,bc Juyoung Yoon*b and Injae Shin*a1.引言分子成像技术能可视化细胞、组织和器官中的（生物）分子和离子，从而得到有关目标分子的生物学信息及其生物功能。
荧光探针如有机染料、荧光蛋白和量子点等已经成为分子成像的重要工具，是因其具有灵敏度高，操作简单，不需要复杂精密的仪器设备等优点1-8。
事实上，荧光探针除了因灵敏度高和选择性好而用于监测生物分子和与生命活动相关的物种外，还广泛用于以时空检测的方式来探测各种生命活动。
在目前大多数情况下，将荧光探针应用于分子成像还处在细胞实验阶段。
然而，对活细胞内的作用对象的成像并不能完全反映出作用目标关于生物功能的全部信息。
与之对比的是，对活体动物的荧光成像能提供更为全面的生物功能信息。
此外，利用与人体具有基因相近的有机体作为载体来研究目标分子生物功能的荧光成像已愈来愈引起人们的关注（图1）。
小鼠是一个较好的动物模型，但对其活体成像需要专业性要求较高的技术和复杂的操作。
而斑马鱼（Danio rerio）已经被证明是一个极有价值的动物模型9。
事实上，斑马鱼用于活体成像有许多优点。
其一，来源简单，无论幼年斑马鱼或成年斑马鱼均能方便获得。
其二，斑马鱼在培育的初期是光透明的，这极大地方便了光学成像技术的运用。
其三，斑马鱼的受精属于体外受精，这使得我们可以方便的观察胚胎发育的各个时期的荧光成像。
此外，斑马鱼与哺乳类动物具有高度的同源性。
基于上述特征，以斑马鱼作模型得到的实验结果可以间接了解目标分子在高级动物中的生物功能。
但是，需要注意的是，由于斑马鱼与哺乳动物毕竟在很大程度上是两类不同的物种，所以由斑马鱼得到的实验结果来外推得到的在哺乳动物上的结果不一定完全可靠。
例如，虽然斑马鱼与哺乳动物在基因上有相似性，但两者在换气和呼吸功能上却截然不同。
斑马鱼综述写作5
Developmental Expression of the Nfe2-Related Factor (Nrf) Transcription Factor Family in the Zebrafish, Danio rerioLarissa M. Williams1,2, Alicia R. Timme-Laragy1, Jared V. Goldstone1, Andrew G. McArthur3, John J. Stegeman1, Roxanna M. Smolowitz4, Mark E. Hahn1*1 Biology Department, Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, United States of America,2 Biology Department, Bates College, Lewiston, Maine, United States of America,3 Andrew McArthur Consulting, Hamilton, Ontario, Canada,4 Department of Biology and Marine Biology, Roger Williams University, Bristol, Rhode Island, United States of AmericaIntroductionThe nuclear factor-erythroid-2 (NFE2)-related factor (NRF) genes encode transcription factors in the Cap’n’Collar basic leucine zipper (CNC-bZIP) gene family. Members of this family include NFE2 [1,2], NRF1 (NFE2L1) [3,4], NRF2 (NFE2L2) [5], and NRF3 (NFE2L3) [6]. (For nomenclature conventions, see footnote 3 of ref [7]..) These transcription factors occur in vertebrates from fish [8] to mammals [9,10], and are known to regulate metabolic [11,12], cell cycle [12-14] and cytoprotective processes [9,13,15-18]. In mice, Nrf proteins have been shown to play important and often essential roles in development. Nfe2-null mice lack circulating platelets [19], the Nrf1 knockout is embryonic lethal [20], Nrf2-null mice are highly sensitive to carcinogens and oxidative stress [21], and the Nrf3knockout does not show abnormalities under normal conditions but is susceptible to lymphoma induced by exposure to carcinogens [22].Although studies in mice have provided important information about the function of Nrf genes, viviparous development makes it difficult to directly observe the role and effects of genes throughout the embryonic period.Zebrafish are a powerful model for studying molecular mechanisms of vertebrate development. Genes and signaling pathways [23,24] and molecular mechanisms of developmental toxicity [25-29] are highly conserved between fish and mammals. Furthermore, due to the whole genome duplication that occurred during early teleost radiation [30-33], zebrafish have duplicated copies (paralogs) that are co-orthologs of many single copy human genes [30,34]. Paralogs that have undergone subfunctionalization provide an opportunity for newmechanistic insights and thus new understanding about the functions of their single human counterpart [32,35]. In zebrafish, there are several paralogs within the nrf gene family, including nrf1a and nrf1b as well as nrf2a and nrf2b [7]. In total, zebrafish have six nrf family genes.In mammals, NFE2 forms a heterodimeric transcription factor with small MAF proteins, regulates expression of globin genes [2,36], and may regulate the oxidative stress response in mature erythrocytes [17]. In zebrafish, nfe2is a single copy gene [8]; its duplicate appears to have been lost [7]. In embryos nfe2 is highly expressed in the intermediate cell mass during erythroid differentiation [8]. Apart from in situ hybridization during development and protein characterization, the expression and role of nfe2in development are largely unexplored.NRF1 in mammals is expressed in many tissues [4]; it plays a role in regulating redox balance in the liver [37] and in regulating genes involved in development [20], oxidative stress [38,39], cytoskeletal organization [40], and the proteasome [41]. In response to UVB damage, mammalian NRF1 regulates nucleotide excision repair and glutathione homeostasis, and may act as a tumor suppressor [13]. The two zebrafish nrf1 paralogs, nrf1a and nrf1b, are syntenic with the duplicated hoxb clusters, similar to the human NRF1 that is near the human HOXB cluster [7]. Prior to the current study, the only information about expression of zebrafish nrf1had been from EST data [38] and a limited assessment by qualitative RT-PCR [7].NRF2 has a broad tissue distribution in mammals and acts as a pleiotropic transcription factor involved in regulating the susceptibility to disease, in mediating the response to xenobiotic exposure and oxidant stress, and in proteome maintenance [42]. In zebrafish, two paralogs (nrf2a and nrf2b) are broadly expressed both in the embryo and adult and apparently have undergone subfunctionalization [7]. The primary role of nrf2a seems to be in the regulation of cytoprotective genes upon oxidative stress [7,9]. Conversely, nrf2b is a negative regulator of embryonic gene expression both basally and under oxidative stress conditions [7].NRF3 has been found to be expressed at high levels in mammalian placenta and the B cell lineage and at lower levels in the heart, lung, kidney, and pancreas [6]. NRF3 may play a protective role in regulating genes responding to oxidative stress, as Nrf3-deficient mice treated with an oxidant suffer acute lung damage [43]. Zebrafish have one nrf3 ortholog, and like nfe2, the duplicated gene appears to have been lost [7]. The expression and function of nrf3in zebrafish was completely unexplored prior to this study.In this paper, we address fundamental aspects of the expression and role of six nrf genes during vertebrate development using the zebrafish as a model system. We show that the expression of nrf genes varies temporally throughout development and that nrf genes are induced following a pro-oxidant exposure and are potentially cross-regulated by gene family members Nrf2a and Nrf2b. We also identify a novel role for nfe2 in the cellular organization of epithelia in the pneumatic duct.Experimental ProceduresFish husbandryZebrafish of the Tupfel/Longfin mutation (TL) wild-type strain were used in all experiments. Adults were maintained and embryos were collected as previously described [44]. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Woods Hole Oceanographic Institution Animal Care and Use Committee (Animal Welfare Assurance Number A3630-01).Cloning and confirmation sequencing of nrf genesIn order to determine nrf cDNA sequences in the TL wild-type strain, and identify strain-specific polymorphisms, nrf cDNAs were amplified with primer pairs (Table 1), using Advantage cDNA Polymerase. The cycling conditions were [94°C, 1 min], [94°C, 30 sec; 68°C, 3 min] for 35 cycles, followed by 68°C for 3 minutes and 10 minutes at 15°C. The PCR products were purified with the GENECLEAN kit (Qbiogene, Quebec, Canada), ligated into pGemT Easy Vector System (Promega, Madison, WI) and constructs were sequenced. Prior to confirmation of in vitro expression, gene products from pGemT were subcloned into pcDNA3.1/Zeo (+) (Invitrogen, Carlsbad, CA). Sequences of clones were deposited into Genbank and have accession numbers JX867113-JX867116.Sampling, Chemical exposure, RNA extraction, and cDNA synthesisEmbryos for Developmental Series.As described in Timme-Laragy et al. [7], 4 pools of 30 staged embryos from a single clutch were reared at low density at 28.5°C in 0.3x Danieau’s and flash frozen in liquid nitrogen at 6, 12, 24, 48, 60, 72, 96, and 120 hpf and stored at -80°C. Hatched and unhatched embryos were collected separately at the 48 and 60 hpf time points. Unfertilized eggs, representing the 0 hpf time point, were manually stripped from 3 females, combined, and flash frozen as 3 separate biological replicates and stored at -80°C. Although it limited our ability to determine individual variation amongst embryos, pooling of embryos was needed to provide sufficient amounts of RNA for measuring expression of multiple genes.Chemical exposure of embryos to tBOOH.Embryos (3 pools of 30 staged embryos) were exposed to tert-butylhydroperoxide (tBOOH). Embryos (96 hpf) were exposed to 0, 0.5 or 0.75 mM tBOOH for 6 hours in 150x20mM glass petri dishes. Following tBOOH exposure, embryos were immediately placed in RNAlater and stored at -80°C.RNA extraction and cDNA synthesis.Total RNA was isolated from pooled embryo samples using RNA STAT-60 (AMS Biotechnology, Abingdon, UK) following the manufacturer’s protocol. cDNA was synthesized from 1 µg of total RNA using random hexamers and the Omniscript cDNA Synthesis Kit (Qiagen, Valencia, CA).Quantitative real-time RT-PCR gene expression profilingUsing the MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad, Hercules, CA), QPCR was conducted with the iQ SYBR Green Supermix (Bio-Rad) on nrf genes. For each sample, duplicate reactions in separate wells were run containing 5 ng of cDNA. The PCR conditions were 95°C for 3.5 minutes followed by 35 cycles of 95°C for 15 seconds and 25 seconds at a gene specific temperature. Gene-specific primers and temperatures are listed in Table 1. Following each run, a melt curve was generated to ensure the amplification of single product. All primers were tested for amplification efficiency using a calibration dilution curve and slope calculation approach [45]. β-actin 1 (actb1) was chosen as a housekeeping gene due to its limited variation in expression with embryonic development and chemical exposure [46]. We confirmed the suitability of this housekeeping gene with the set of samples used in this study. Expression of genes wasanalyzed using the comparative ∆∆CT method [47].Statistical analysis of QPCR dataDevelopmental Series. Data were analyzed with MicrosoftExcel using the ∆∆CTmethod to calculate fold change. Valuesare presented as mean ± SEM, and N is defined as the numberof pools of embryos. Relative expression was normalized to the120 hpf value within a gene. As described in Timme-Laragy etal. [7], Statview for Windows (version 5.0.1; SAS Institute,Cary, NC) was used to determine differences in expression dueto hatching state. Data were log-normalized and six statisticaloutliers were removed from the nrf2b development series (onedata point from each of six time points: 0, 6, 24, 48(unhatched), 48 (hatched), and 96 hpf). If an ANOVA yieldedsignificance (p< 0.05), Fisher's protected least significantdifferences test was used as a post hoc test with Bonferronicorrection.Chemical Exposure and Nrf2a and Nrf2b crosstalk. Datawere analyzed with Statview for Windows (version 5.0.1; SASInstitute, Cary, NC) and presented as mean ± SEM where N isdefined as the number of pools of embryos. Significance inANOVA was determined with a p≤ 0.05. When significancewas yielded, Fisher’s protected least-significant differences testTable 1. Primers used for gene amplification, cloning, and qPCR of nrf genes.Gene Primer sequences (5’-3’)Amplicon size T (°C)RefR-GGTGAACTATCACTTTAATCAAACATnrf1a F-CAGTTCGCACGCCCTTATTTACTGAC237668-R-GCTGATGGACTTAACAGCAGACAGnrf1b F-CGTAACCTAATTTGGTTTGACG288568-R-GTCCTCCTTGACTTCCCATATCnrf3F-AAATTGAGCAGTTGCTCCCCTCC234668-R-CTCACCTCAAAGATAAAACTCACCnfe2-qPCR F-CAGAGTTTGAGGAACCCAATGAG12757-R-CACAAGTGGCTGGAATGGATTCnrf1a-qPCR F-CCAGAGTTGACAGGTCCTGG15864-R-CATAACCTGTGATTCCATGATAGACnrf1b-qPCR F-GCAGGACATGGAGGTGAACAATACG12066-R-GGATCGTGGGAGCCCAAAATTTCCnrf2a-qPCR F-GAGCGGGAGAAATCACACAGAATG8265[7] R-CAGGAGCTGCATGCACTCATCGnrf2b-qPCR F-GGCAGAGGGAGGAGGAGACCAT26968[7] R-AAACAGCAGGGCAGACAACAAGGnrf3-qPCR F-GCATGAGGATTTAGTGGTTAGTGG10864-R-GGAGTCAAAATCATCAAAGTCAGactb1-qPCR F-CAACAGAGAGAAGATGACACAGATCA14065[87] R-GTCACACCATCACCAGAGTCCATCACPrimer sequences are written 5’-3’ and amplicon size and melting temperature (T) are shown. For primers used previously, a reference is cited. Accession numbers for sequences newly reported here are: nrf1a (JX867114), nrf1b (JX867116), nrf3 (JX867113), nfe2 (JX867115).doi: 10.1371/journal.pone.0079574.t001was used as a post hoc test with Bonferroni correction. Relative expression was normalized to the water control for tBOOH. For Nrf2a and Nrf2b morpholino data, relative expression of each gene was normalized to the water control with control morpholino.Microarray gene expression profilingZebrafish embryo rearing, sampling, and microarray analysis were performed as previously reported [29]. Briefly, four replicate pools of 100 embryos were examined at 3, 6, 12, 24, 36, and 48 hpf. Agilent 4 × 44k DNA gene expression microarrays (part #GPL11077, WHOI Zebrafish 44k v1.0 custom-commercial Chemical Defensome array, Agilent Technologies, Santa Clara, CA), which included custom probes to ensure adequate coverage of the chemical defensome [29], were used to examine gene expression levels. Single color microarray data (Cy3) was normalized using the non-linear scaling method of Schadt et al. [48], where saturated probes and probes not above background in all replicated were removed. Because standard ANOVA is not appropriate for these data due to auto-correlation between time points, we used Bayesian Estimation of Temporal Regulation (BETR; [49]) to analyze the developmental time series. Normalized Cy3 values for each probe were log transformed, median-centered, and analyzed using BETR relative to 3 hpf. Normalized average Cy3 signals were compared for genes of interest among time points. All probes reported here had significant differential expression among sampling times. The microarray data have been deposited into the Gene Expression Omnibus (GEO) database with NCBI GEO accession number GSE24840.In situ hybridizationIn situ hybridization of nrf3 on whole embryos was performed using established procedures [50,51]. Sense and anti-sense nrf3 RNA probes were created by restriction digest of the nrf3 construct (in pGemT) with XhoI, yielding a 1080 bp sense probe and 1328 bp anti-sense probe. The nrf3 probe covers a 1240-bp region of the 3’ end of the cDNA and includes 87 base pairs of the 3’ UTR. In designing the probe, we carefully considered possible sequence identity with other nrf transcripts. Nucleotide sequence identities between nrf3and other nrf transcripts in the region covered by the probe were as follows: 45% with nfe2, 47% with nrf1a, 51% with nrf1b, 49% with nrf2a, and 45% with nrf2b. The overlap between the nrf3 probe and other nrf transcripts was typically interspersed throughout the sequence with no long runs of overlap (mainly, 4-5 nt at a time). We also conducted a Blast search using the nrf3 probe against the zebrafish genome; no significant overlap was found other than the nrf3 gene itself. Probes were labeled with digoxigenin-UTP by in vitro transcription (Roche San Francisco, CA) with either T7 (sense) or SP6 (anti-sense) polymerase. Pre-hybridization was carried out for 2 hours at 65°C. Hybridization was carried out overnight with 300 ng of probe per pool of seven embryos at a concentration of approximately 1.5 µg/mL. Embryos were blocked for 4 hours at room temperature and incubated with anti-digoxigenin alkaline phosphatase conjugated antibody (Roche, San Francisco, CA)at 4°C overnight. Embryos were imaged by light microscopy (Axio Observer.A1, Carl Zeiss, Oberkochen, Germany).Morpholino oligonucleotidesMorpholino antisense oligonucleotides (MO) were designed to block initiation of translation of zebrafish nfe2, nrf1a, nrf1b and nrf3 and obtained from Gene Tools, LLC (Philomath, OR). The first nfe2MO (AGTTCCTGCCAGGCCAAGTCCATCT) complements two residues of the 5’UTR, the start codon (underlined), and 20 residues of the coding region. A second, non-overlapping nfe2MO (AACGATGTGTCCGTAATCCAGTGAC) complements 25 residues of the 5’UTR and targets a region whose 3’ end is 15 residues upstream of the sequence targeted by the first nfe2 morpholino. The nrf1a MO (ATGGCCCAAACCATCACCGGCAGCA) complements 11 residues of the 5’UTR, the start codon (underlined), and 11 residues of the coding region. The nrf1b MO (AATCACGCAAACAAACGTCAAACCA) complements 25 residues of the 5’UTR and its 3’ end is located 34 residues upstream of the start codon. The nrf3MO (TTTTAACCTCAGGAGGCTTAAACGA) complements 25 of the 5’UTR and its 3’ end is located 14 residues upstream of the start codon. The standard control-MO from Gene Tools was also used (CCTCTTACCTCAGTTACAATTTATA). All MO were tagged at the 3’ end with fluorescein in order to detect successful injection and incorporation of MO into embryos. MOs targeting nrf2a and nrf2b are previously described [7]. Confirmation of MO translation inhibition by in vitro protein synthesisTo confirm the efficacy of morpholinos to inhibit translation of Nrf proteins, the TnT T7 Quick Coupled Reticulocyte Lysate System (Promega, Madison, WI) was used to synthesize [35S]methionine-labeled zebrafish NF-E2, Nrf1a, Nrf1b and Nrf3 protein as per manufacturer’s protocols and as described previously [52]. Briefly, the TnT reaction included 1 μl of [35S]methionine (> 1000 Ci/mmol at 10 mCi/ml) and 2 μl of the respective nrf cDNA in pcDNA3.1/Zeo (+) (0.5 μg/μl) in a final volume of 25 μl with H2O. For reactions containing MOs, 0.5 μl of a 25 μM stock of the appropriate MO was added, resulting in a final concentration of 500 nM. Labeled protein products were resolved by SDS-PAGE, dried on Whatman filter paper, and visualized on film. To quantify the reduction in protein products, gel fragments containing a single protein band were excised and placed into 7 mL glass scintillation vials containing 5mL ScintiVerse II cocktail (Fisher Scientific, Pittsburg, PA). Counts were carried out for 5 minutes on a Beckman Coulter LS6500 Multi-purpose Scintillation counter (Brea, CA) and background was subtracted.Microinjection of zebrafish embryos with morpholinos Zebrafish embryos at the two- to four-cell stage were injected with 2.5-5 nL of a 0.1 mM solution of MO targeting nfe2, nrf1a, nrf1b, nrf2a, nrf2b, nrf3, or a control-MO, using a Narishige IM-200 microinjector (Tokyo, Japan). Injection volumes were calibrated as described previously [52]. The injections resulted in delivery of MO amounts ranging from 2-4 ng. At six to ninehpf, embryos were sorted and screened for successful fertilization and fluorescence. When no developmental phenotypes were observed, the concentration of the morpholino stock was increased to 0.15 mM, keeping the injection volume constant. MO-injected embryos were held in 0.3x Danieu’s solution. At 4 dpf, swim bladder inflation was scored as described earlier [53].Histological analysis of the swim bladderFour-day-old larvae from embryos injected with control morpholino (swim bladder inflated) or nfe2morpholino (swim bladder not inflated) were fixed in 4% formaldehyde in 1x phosphate buffer, dehydrated in ethanol and stored in 75% ethanol until embedding. Larvae were sent to Environmental Pathology Laboratories (Sterling, VA) where they were embedded laterally into either Technovit 7100 (Heraeus Kulzer, Hanau, Germany) or paraffin. Sagittal sections were made serially every 2 µm. Sections were mounted on superfrost glass slides and stained with hematoxylin and eosin. Histopathology was conducted by an Olympus BX 40 with an Olympus DP25 camera system.Analysis of blood flowTransgenic gata1dsRED embryos [54] (15 individuals per treatment) were injected with control or nfe2-MOs at one- to four-cell stages. As an index of blood flow, the number of red blood cells passing through the mesencephalic vein (MsV) per 15 s was determined by orienting 48 hpf zebrafish embryos laterally as described by Kubota et al. [55]. Blood flow was recorded with an AxioCam MRc5 camera on a Zeiss Axiovert 200 inverted microscope and analysis (counting of red blood cells) was performed using ImageJ (/ij/).Analysis of otic vesiclesAB embryos were injected at one- to four-cell stages with control-MO or nfe2-MO-1. At 30, 48 and 72 hpf, otic vesicles were imaged with an AxioCam MRc5 camera using a Zeiss Axiovert 200 inverted microscope.In silico Promoter Analysis and motif searchesTo determine whether Nrf2a or Nrf2b are directly involved in the transcriptional regulation of other nrf family members, in silico promoter analysis was carried out for AREs and XREs using a fuzzy search algorithm, fuzznuc [56]. 10,000 base pairs upstream of the start site and the entire length of the gene were examined for putative zebrafish AREs (TGA(G/C)nnnTC [57]) and XREs (KNGCGTG [58]). Additional searches were carried out for Nfe2 binding sites [59] determined from ChIP-Seq studies. The zebrafish genome was searched for Nfe2 binding sites in the 10 kb upstream of the start codon of all known genes using a position-specific scoring matrix (PSSM) based on the empirical ChIP-Seq results of Wang et al [59] and the FIMO algorithm of the MEME/MAST package [60](p < 0.0001), with correction for background nucleotide frequencies of complete nuclear genome sequences.ResultsDevelopmental expression profilingTo begin to understand the role of the nrf genes during development, their expression was profiled from 0 to 120 hpf using qRT-PCR (Figure 1). Because the data were normalized within each gene, expression values can be compared at different times for each gene but not across genes. nfe2 transcripts were maternally deposited, but levels fell substantially at 6 hpf and did not increase again until 48 hpf. Expression remained relatively constant from 48 to 60 hpf and then decreased steadily until 120 hpf. Like nfe2, nrf3was maternally deposited and decreased by 6 hpf, but transcript levels increased again at 12 hpf. Expression decreased between 12 and 48 hpf, but then remained steady. nrf1a was expressed from 0-24 hpf but levels were reduced from 48-72 hpf, after which expression increased at 96 and 120 hpf. Conversely, nrf1b expression peaked between 12 and 24 hpf and then declined and remained relatively low from 48 to 120 hpf. Hatching did not have a significant effect on expression of nfe2, nrf3, nrf1a or nrf1b. For comparison, Figure 1 also includes data for expression of nfr2a and nrf2b, originally presented (in a slightly different format) by Timme-Laragy et al.[7]. These results showed that nrf2a expression increased steadily during development. The expression of nrf2b also increased through 48 hpf, but there was a significant effect of hatching on nrf2b expression, when expression decreased in hatched embryos compared to unhatched embryos of the same developmental age.Since molecule numbers were not available from the qRT-PCR data and data were normalized within each gene, a microarray was used to determine the expression of nrf genes relative to each other during the first 48 hours of development (Figure 2). Microarray and QPCR analyses were performed on samples obtained from independent experiments. The microarray data did not include a 0 hpf time point, so maternal transcript levels were not determined with this method. Gene expression profiles determined by QPCR and microarray for nfe2, nrf1a, nrf2a, and nrf2b displayed similar developmental patterns. The developmental patterns of nrf3expression measured by qPCR and microarray were similar, except that the pattern at 6 and 12 hpf was reversed. For nrf1b, the peak at 12 hpf measured by QPCR was not evident in the microarray data.Normalized Cy3 levels for nfe2, nrf1a, nrf1b, nrf2a, and nrf2b ranged from 100 to 1,800, whereas values for nrf3 ranged from 80,000 at 3 hpf to approximately 20,000 at 48 hpf (Figure 2). The nrf gene with lowest expression was nrf1a followed by nrf1b. Their average expression values were about half that of nfe2. The expression of nrf2a was up to10-fold greater than that of all other nrf genes except nrf3.Localization of nrf3 expression with ISHWe used in situ hybridization to examine the spatial expression of nrf3, the nrf gene most highly expressed during development; analysis was carried out in 12-hour intervals from 24 to 72 hpf. In situ gene hybridization results for nfe2, nrf1a, and nrf2a have been previously reported [8,61,62]. The mostprominent expression of nrf3 occurred at 24 and 36 hpf;expression was detected in the fore-, mid- and hindbrain as well as in the pectoral fin buds (Figures 3A, 3B, 3D, 3E). The high degree of staining seen is consistent with the relatively high level of nrf3 expression seen in the array and qRT-PCR data. At 48 hpf and 60 hpf, expression still was seen in the pectoral fin buds, and was less prominent in the forebrain andmore concentrated in both the mid- and hindbrain (Figures 3G,Figure 1. The expression of the nrf gene family during development as measured by quantitative real-time PCR. Black lines indicate expression pre-hatch and grey lines indicate expression post-hatch. Values were normalized to the 120 hpf time-point and β-actin 1 (actb1) was used as the housekeeping gene. Data are presented as the mean ± S.E.M. (error bars), and N = 4 pools of 30 embryos. Differences in expression between hatching state were assessed using ANOVA followed by Fisher's PLSD (*, p ≤0.05). Data for expression of nrf2a and nrf2b are from Timme-Laragy et al. [7] and are used with permission of the American Society for Biochemistry and Molecular Biology. Previously these data were presented as molecule number. Here we have normalized the data to β-actin 1 expression and to the 120 hpf time point and re-plotted for comparison to the new data on expression of other nrf family genes. Dashed lines indicate the value of 1.0 from the 120 hpf time point.doi: 10.1371/journal.pone.0079574.g0013H, 3J, 3K). Expression was also seen in the branchiogenic primordia. Expression at 72 hpf was almost exclusively in the mid/hindbrain junction (Figure 3M, 3N). There was no signal in sense controls at any time point assayed (Figures 3C, 3F, 3I,3L, 3O).Effect of nrf gene knockdown on developmentOne approach to identify the importance of nrf genes in developmental processes is to knock down their expression in the embryo. The in vitro translation-blocking efficiency of nrf morpholinos ranged from 65 to 68%, with the exception of morpholino 2 for nfe2 (22%; Figure 4). To determine whether any morphological abnormalities were associated with the reduction of Nrf proteins, embryos were injected with morpholinos and observed every 12 hours until 120 hpf. No gross morphological abnormalities were observed for control MO, nrf1a -MO, nrf1b -MO, a combination of nrf1a -MO and nrf1b -MO, or nrf3-MO. Additional experiments were conducted using higher concentration of morpholino (0.15 mM). However,at this concentration off-target effects were observed. Off-targeteffects observed in more than 50% of the MO-injected embryos included hatching gland deformities, pericardial edema, and bent tail post-hatching.In contrast to the lack of specific phenotypes in embryos injected with MOs targeting nrf1a, nrf1b , or nrf3, injection of an nfe2-MO (0.1 mM) resulted in the failure of the swim bladder to inflate at 96 hpf in 95% of the injected embryos (Figure 5B versus 5A) (Table 2). The specificity of this effect was confirmed using a second, non-overlapping nfe2 MO that also targeted the region surrounding the translation start site (Table 2).Histological analysis of larvae treated with control MO showed the normal pneumatic duct connection between the esophagus and the swim bladder (Figure 5C,D). Epithelial cells along the duct were orderly and cell morphology progressed from columnar at the entrance to the duct to cuboidal to squamous at the entrance to the swim bladder. In larvae in which Nfe2 had been knocked down, the pneumatic duct connection was intact but the number of epithelial cells and the regular progression from columnar to squamous was disrupted (Figure 5E). In the most severe example, the epithelialcellsFigure 2. The expression of the nrf gene family during development as measured by microarray analysis. Data are presented as normalized Cy3 levels, with average values for nfe2, nrf1a , nrf1b , nrf2a , and nrf2b displayed on the primary (left) y-axis. Values for nrf3 are displayed on the secondary (right) y-axis. Each time point represents the average normalized value ±SD of 4 replicates, each from 100 embryos. The Agilent probes from which these data were obtained are: nfe2 (A_15_P109440), nrf1a (A_15_P110831), nrf1b (A_15_P116909), nrf2a (CUST_121_PI358351581), nrf2b (CUST_122_PI358351581), nrf3(A_15_P116878). An additional probe for nrf2a (A_15_P109504) was present on the array and produced results very similar to those shown here for CUST_121_PI358351581. The microarray data have been deposited with NCBI GEO accession number GSE24840.doi: 10.1371/journal.pone.0079574.g002。
模式生物斑马鱼在药物毒性研究中的应用进展综述
2019.26科学技术创新模式生物斑马鱼在药物毒性研究中的应用进展综述钱星宇（江苏省徐州市第三中学，江苏徐州221000）模式生物是指在研究生命现象的过程中，长期重复使用的非人类物种。
它们通常体积小、结构简单、繁殖快、后代多、成本低、基因组小，可以代表一个大的群体。
大鼠、小鼠、爪蟾、河豚、斑马鱼、海胆、果蝇、秀丽隐杆线虫、线虫、玉米、拟南芥、盐芥、流感嗜血杆菌、酿酒酵母和大肠杆菌是常用的模式生物[1]。
模式生物在生物学研究中有很多应用。
目前，主要应用于人类基因组研究、发育的基因调控的研究、遗传学研究、动物行为范式分析、学习记忆与某些认知行为的研究、衰老与长寿研究、各类神经疾病的研究、帕金森氏病研究、药物生理机制研究、药物有效性研究、药物成瘾和酒精中毒研究、药物毒性研究等方面。
斑马鱼是药物毒性研究中的常见模式生物，在药物的发育毒性与致畸性、心血管毒性、肝毒性、行为毒性和肾毒性等一系列评价实验当中应用较广。
本文就模式生物斑马鱼在药物毒性研究中的应用进展进行综述。
1关于斑马鱼斑马鱼是鲤科的一种骨质小鱼，俗称蓝条纹鱼、条纹鱼和斑马坦尼鱼。
它是一种产于印度、孟加拉和其他水域的小型热带鱼[2]。
斑马鱼具有体积小、饲养成本低、繁殖周期短、产卵率高、体外受精、药物用量少、卵大、易于观察、与人类基因同源性高达87%等优点[3]，是理想的模式生物。
可用于发育毒理学、病理毒理学、药物毒理学[4]、正反遗传学、药物研究与开发以及人类疾病研究。
在药物毒性研究的多个领域中，斑马鱼都有着广泛应用，如药物的发育毒性与致畸性、心血管毒性、肝毒性、肾毒性和行为毒性等[5]。
2斑马鱼在药物毒性研究中的应用2.1斑马鱼在药物的发育毒性与致畸性研究中的应用以斑马鱼作为模式生物的药物发育毒性实验的主要内容是根据斑马鱼胚胎的畸形率和死亡率等指标，评价测试药物的发育毒性，以指导临床合理用药，对开发新药也有一定的意义。
利用斑马鱼进行药物发育毒性的评价，具有操作简易、实验周期短等优点，只要短短的1~2天，就可以评价药物是否具有毒性。
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》范文
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》篇一一、引言近年来，BUVSs（一种新型化学物质）在环境中的广泛存在引起了科学家的关注。
由于其在生物体内的潜在影响，研究其代谢特征及毒性效应变得尤为重要。
斑马鱼作为一种常用的模式生物，具有与人类相似的生理和代谢机制，因此常被用于研究化学物质的生物效应。
本文旨在探讨BUVSs在斑马鱼体内的代谢特征及其毒性效应。
二、BUVSs在斑马鱼体内的代谢特征1. 吸收与分布BUVSs进入斑马鱼体内后，通过消化系统被吸收，随后分布于各组织器官。
在肝脏和肾脏中，BUVSs的浓度较高，这可能与肝脏和肾脏对BUVSs的代谢和排泄有关。
2. 代谢途径BUVSs在斑马鱼体内的代谢途径主要包括氧化、还原、水解等反应。
其中，氧化反应是BUVSs的主要代谢途径之一，产生的代谢产物可能具有不同的生物活性。
此外，BUVSs也可能与体内的其他物质发生结合反应，形成新的化合物。
3. 代谢动力学BUVSs在斑马鱼体内的代谢动力学特征包括吸收、分布、代谢和排泄等过程。
通过测定BUVSs在斑马鱼体内的浓度随时间的变化，可以了解其代谢动力学特征，为评估其潜在风险提供依据。
三、BUVSs对斑马鱼的毒性效应1. 对生长与发育的影响BUVSs对斑马鱼的生长与发育具有一定的抑制作用。
长期暴露于BUVSs环境下，斑马鱼的生长速度减慢，体型变小。
此外，BUVSs还可能影响斑马鱼的繁殖能力，导致后代数量减少。
2. 对生理功能的影响BUVSs可能影响斑马鱼的生理功能，如影响神经系统、内分泌系统和免疫系统等。
具体表现为行为异常、激素水平紊乱和免疫反应等。
此外，BUVSs还可能对斑马鱼的感官器官造成损害，如影响视觉和嗅觉等。
3. 对基因表达的影响研究表明，BUVSs可能影响斑马鱼的基因表达，导致基因突变和基因组不稳定。
这可能对斑马鱼的生存和繁衍产生长期影响，并可能对人类健康构成潜在风险。
四、结论通过对BUVSs在斑马鱼体内的代谢特征及其毒性效应的研究，我们了解到BUVSs在生物体内的吸收、分布、代谢和排泄等过程。
斑马鱼描写作文
斑马鱼描写作文“哇，这小鱼也太漂亮了吧！”我和小伙伴一起趴在水族馆的玻璃前，眼睛直勾勾地盯着里面游来游去的斑马鱼。
周末，我和几个好朋友相约来到水族馆。
一走进这里，仿佛进入了一个神奇的水下世界。
五彩斑斓的灯光打在巨大的玻璃缸上，各种各样的鱼儿在水中欢快地游弋着。
我们几个像好奇宝宝一样，一会儿跑到这个缸前看看，一会儿又跑到那个缸前指指点点。
当我们来到斑马鱼的鱼缸前时，一下子就被它们吸引住了。
这些斑马鱼就像一群小精灵，身上的条纹黑白相间，整齐而又醒目。
它们在水中灵活地穿梭着，时而快速游动，时而停下来摆摆尾巴，那模样真是可爱极了。
“嘿，你说这些斑马鱼像不像马路上的斑马线呀？”一个小伙伴笑着问道。
“还真挺像呢！”我回应道。
“它们游得好自在呀，要是我们也能像它们一样无忧无虑就好了。
”另一个小伙伴感叹道。
看着这些斑马鱼，我不禁陷入了沉思。
它们在这个小小的鱼缸里，却依然充满活力，尽情地享受着自己的生活。
而我们呢，有时候总是为了一些小事烦恼，为了考试成绩担忧，为了和父母的矛盾不开心。
和这些斑马鱼比起来，我们是不是有点太“矫情”了呢？这些斑马鱼就像是生活中的一面镜子，让我看到了自己的不足。
它们用自己的行动告诉我们，无论生活在什么样的环境里，都要保持乐观积极的心态，勇敢地面对一切。
我们在水族馆里待了很久，直到闭馆才恋恋不舍地离开。
回家的路上，我的脑海里还一直浮现着那些可爱的斑马鱼。
它们虽然渺小，却有着强大的生命力和乐观的精神。
我想，以后我也要像斑马鱼一样，无论遇到什么困难，都要勇敢地向前游，永不放弃。
本人原创声明：创作不易，请体谅作者的辛苦工作，谢谢!。
华中农大：斑马鱼养殖综述
斑马鱼(Danio rerio)养殖综述Christian Lawrence布莱根妇女医院卡帕研究实验室马萨诸塞州美国收稿日期：2007年7月16日；改修日期：2007年8月24日；接受日期：2007年8月25日。
摘要：斑马鱼(Danio rerio)是脊椎动物研究的模式生物。
斑马鱼的优良性状有利于研究人类疾病的发生与传播。
较强的繁殖力、短小的体型、繁殖周期短、胚胎早期光学透明性强等特性使得斑马鱼成为许多研究者研究其他项目青睐的对象，包括研究动物行为、鱼类生理机能和水产养殖毒性试验。
尽管如此，科学严谨的斑马鱼养殖技术还是很不发达。
尽管斑马鱼有很大的需求量，包括直接或者间接养殖。
斑马鱼的养殖缺乏应用养殖标准。
本文试图将已有的关于斑马鱼生物学和养殖的科学信息进行综述，可以用来提高这一重要的模型动物使用检索的效率。
本研究还强调了该领域还需要进一步研究的内容。
关键词：斑马鱼；Danio rerio；饲养；水产养殖；管理目录：1、引言 (2)2、斑马鱼自然生活史 (3)2.1、分布和栖息地 (3)2.2、生殖和行为 (3)2.3、寿命 (5)2.4、食物 (5)3、斑马鱼养殖 (5)3.1、水化学参数 (5)3.1.1、温度 (6)3.1.1、pH (6)3.1.3、硬度 (7)3.1.4、盐度 (7)3.1.5、溶解氧 (8)3.1.6、含氮废弃物 (8)4、营养、食物、投喂方式 (8)4.1、营养需求 (8)4.2、食物 (9)4.3、投喂 (11)5、繁殖和育种技术 (11)5.1、繁殖 (11)5.2、育种技术 (12)5.3、产卵效率 (13)6、幼鱼饲养 (13)6.1、幼鱼生物学 (13)6.2、食物和营养 (13)6.3、水质 (14)6.4、生长率和存活率 (15)7、成鱼培育 (15)7.1、放养密度 (15)7.2、遗传育种 (16)8、结语 (16)鸣谢 (16)参考文献 (16)1、引言在过去的二十年间，斑马鱼(Danio rerio)广泛作为遗传学和发育学研究的优良模式生物(Fishman,2001)，最近斑马鱼还应用于的人类疾病和治疗药物筛选的研究当中(Penberthy et al.,2002;Sumanasa and Lin, 2004)。
斑马鱼简介
它是发育生物学家的得力助手，是医学研究的后起之秀，是环境监测的哨兵，是指示兵，是药物研发的新宠，它就是脊椎类模式动物明星斑马鱼（zebrafish，Danio rario）。
一、斑马鱼基本特点：原产地：热带淡水鱼，原产于喜马拉雅山南麓的印度、巴基斯坦、孟加拉和尼泊尔等南亚国家。
成鱼体长3-100px，略呈纺锤形，头小而稍尖，吻较短，身躯玲珑而纤细，因其体侧具有像斑马一样纵向的暗蓝色与银色相间的条纹而得名。
http://www.mun.ca/biology/desmid/brian/BIOL3530/DEVO_03/ch03f09.jpg 斑马鱼是体外受精发育，胚体透明。
胚胎发育快，受精后3天左右孵化出膜，天左右开口进食，约3个月达到性成熟，寿命可达2年以上。
斑马鱼可常年产卵，繁殖周期3-4天，一对成年斑马鱼每次可产卵200-300枚，受精率通常在70%以上。
养殖温度一般在23-31℃，15℃左右仍可存活，最佳养殖温度在25-28摄氏度之间，pH值在6.8~7.8，硬度在2~6之间。
二、斑马鱼作为模式生物的起源和发展1938年，美国布朗大学Roosen-Rung教授首次报道斑马鱼发育形态学研究成果。
1950年代，美国罗格斯大学K. Kenneth Hisaoka教授首次报道斑马鱼毒理学研究成果。
1972年，美国俄勒冈大学George Streisinge教授开始斑马鱼发育生物学研究和模式动物建立工作。
1989年，美国俄勒冈大学Monte Westerfield教授出版斑马鱼研究圣经The Zebrafish Book第一版。
1998年，首个斑马鱼模式生物数据库ZFIN成立（）。
1998年，全球第一家斑马鱼药物研发服务外包公司成立。
2004年，斑马鱼国际资源中心（ZIRC）在俄勒冈大学成立。
2007年，美国环境保护署（EPA）将斑马鱼技术列入其转化毒理学研究项目ToxCast TM。
2009年，斑马鱼药物毒理学评价技术首次通过FDA和EMA的GLP认证，这标志着斑马鱼模型的安全药理学和毒理学评价结果可以作为正式材料纳入临床试验申报资料。
我最喜欢的小动物斑马鱼作文
我最喜欢的小动物斑马鱼作文
我家的鱼缸里养了几条小斑马鱼，它们非常可爱，我很喜欢它们。
斑马鱼的身体是紫色的，像一条美丽的裙子。
它们有一双黑色的眼睛，像两颗黑色的宝石。
还有一张圆圆的小嘴，嘴巴下面有六个又小又尖的牙齿，像两把锋利的宝剑。
斑马鱼的尾巴像一把伞，能为斑马鱼遮风挡雨。
斑马鱼喜欢吃鱼食，但是它们可不喜欢吃面包虫、小虾子等。
所以我经常喂它们吃小鱼虫。
它们吃饱了就会在水里游来游去，好像在说：“真好吃！”
斑马鱼游泳时非常好看，因为它能摆动尾巴划水前进。
我还发现：斑马鱼走路时是用尾巴摆动来移动身体的，这一点和人走路很像啊！
斑马鱼喜欢跟人玩游戏。
它们有时躲在水草后面，有时像个杂技演员在表演“顶球”：它们一会儿把尾巴举得高高的，一会儿把尾巴弯成一个圆圈，一会儿把尾巴展开像个“降落伞”……它们玩得真开心啊！
我还发现斑马鱼睡觉时也很有趣。
它会先把身体贴着水面睡觉，等自己睡着了以后再把尾巴翻过来像个“小船”似的漂浮在水面上。
—— 1 —1 —。
斑马鱼研究报告
斑马鱼研究报告（Zebrafish,Danio rerio）目录一﹑斑马鱼二﹑斑马鱼基因与人类极为相似三﹑利用斑马鱼作为模式动物四﹑斑马鱼的心生五﹑突变斑马鱼及转基因斑马鱼表現类似人类疾病六﹑斑马鱼胚胎研究七﹑研究者在活的斑马鱼身上直接观看器官的发育与病变八﹑解开人与体內活菌共生秘密九﹑斑马鱼胚胎基因有助研究癌症、阿滋海默和帕金森症十﹑视网膜有自我修复潜能十一﹑培育变种斑马鱼可望用于人脸整型十二﹑基因決定人类肤色十三﹑斑马鱼的运动情形肌肉的发育及肌肉与运动神经的调控机制十四﹑细胞如何储存脂肪有助減肥新疗法十五﹑类固醇荷尔蒙的功能与调控十六﹑中神经细胞形成之分子调控机制十七﹑斑马鱼基因调控网路十八﹑干扰斑马鱼基因的技术十九﹑培育环保转基因斑马鱼二十﹑改造斑马鱼基因來试水质二十一﹑利用斑马鱼作为人类疾病模型及药物节选二十二﹑药物节选二十三﹑抗癌药物节选斑马鱼原产于东印度恒河流域，亦分布于巴基斯坦、尼泊尔、缅甸。
斑马鱼成鱼体长约4～5公分，体呈纺锤形，稍侧扁。
体侧从头至尾布满多条蓝色条纹，酷似斑马，故得名斑马鱼。
1.斑马鱼是研究发育生物学的新兴模式动物。
2.斑马鱼由于具有饲育容易、胚胎透明、体外受精、突变种多、遗传学工具成熟等诸多优点，近年来已成为研究脊椎动物发育与人类遗传疾病的新兴模式动物。
3.与其他脊椎动物相较下，斑马鱼最大的优点就是具有多达6000多种的遗传突变种，这些突变种的建立大致上是利用X射线、ENU或反转录病毒的感染造成基因组的突变，之后再经由多次的子代筛选所得。
4.突变种的表征包含如胚层分化，器官发育，生理调适与行为表现等多方面，所以可提供研究人员极佳的正向遗传学材料来进行发育机制上的研究。
5.在斑马鱼系统中也开发出阻断基因功能的工具-Morpholino，可快速以逆向遗传学手法来验证基因的功能。
所以正向遗传学与逆向遗传学的巧妙利用，可以正确推导出斑马鱼遗传发育途径，也是目前斑马鱼成为研究人类疾病新兴模式动物的主要原因。
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》范文
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》篇一一、引言近年来，BUVSs（一种新型化学物质）在环境中的广泛存在引起了科学家的关注。
由于其在生物体内的代谢特征及其潜在的毒性效应，对BUVSs的研究变得尤为重要。
本文以斑马鱼为研究对象，探讨BUVSs在斑马鱼体内的代谢特征及其毒性效应，以期为评估BUVSs的环境风险提供科学依据。
二、材料与方法1. 材料（1）BUVSs样品：本实验所使用的BUVSs样品来源于环境样本。
（2）斑马鱼：本实验选用健康的成年斑马鱼作为研究对象。
2. 方法（1）代谢特征研究通过在斑马鱼体内注射BUVSs，观察其在斑马鱼体内的吸收、分布、代谢及排泄等过程。
采用现代生物分析技术，如高效液相色谱、质谱等，对BUVSs的代谢产物进行定性、定量分析。
（2）毒性效应研究采用一系列生物学指标，如生长速率、行为变化、生理生化指标等，观察BUVSs对斑马鱼的毒性效应。
同时，通过组织学和细胞学方法，观察BUVSs对斑马鱼体内器官和细胞的影响。
三、BUVSs在斑马鱼体内的代谢特征经过研究，我们发现BUVSs在斑马鱼体内具有良好的吸收性，能够在短时间内达到较高的血药浓度。
BUVSs在斑马鱼体内的分布广泛，主要分布在肝脏、肾脏等重要器官。
在代谢过程中，BUVSs经过一系列生物转化，生成多种代谢产物。
这些代谢产物通过尿液和胆汁等途径排出体外。
四、BUVSs对斑马鱼的毒性效应1. 生长速率BUVSs对斑马鱼的生长速率具有显著影响。
暴露于BUVSs 的斑马鱼生长速度明显减慢，体长和体重增长受阻。
2. 行为变化BUVSs可导致斑马鱼行为发生改变。
暴露于BUVSs的斑马鱼活动减少，游动速度变慢，回避反应减弱。
这些行为变化可能与BUVSs对斑马鱼神经系统的影响有关。
3. 生理生化指标BUVSs可引起斑马鱼体内多种生理生化指标的变化。
例如，暴露于BUVSs的斑马鱼血液中某些酶的活性发生变化，表明BUVSs可能对斑马鱼的肝脏、肾脏等器官造成损伤。
文献综述-斑马鱼及其应用
斑马鱼及其研究应用作者：杜颖指导老师：张源淑摘要：斑马鱼作为一种新兴的重要模式动物之一，体外受精、胚胎透明，因此可在显微镜下直接观察发育过程及检测药物引起的内脏组织变化，在生命科学领域中应用前景十分广阔。
斑马鱼体型小,适合高通量研究,还具有生长繁殖周期短及其与人类高度相似的基因组等优点，已经广泛用于人类疾病模型的建立、新药研发和药物的筛选，此外，斑马鱼还被应用于毒理学、发育生物学和遗传学等的研究。
因为斑马鱼对污染物反应灵敏，现已用于监测环境污染物及污水检测。
本文主要从几个方面对斑马鱼的研究进展进行了整理和归纳。
关键字：斑马鱼模式动物科学研究发育感染药物Zebrafish and Its ApplicationAbstract:Zebrafish as an important model animal emerging, its in vitro fertilization, transparent embryo, internal organs can be directly observed during the development and testing organ change caused by drugs under the microscope, has very broad application prospects in the field of life sciences. zebrafish also has a live, high-throughput, growth and short reproduction cycles and highly similar to the human genome, etc., it is widely used in modeling human diseases, drug screening, and secondly, zebrafish also is applied in toxicology research, developmental biology and genetics, etc.. Because of its sensitivity, it has been used to monitor environmental pollutants and water testing.This paper mainly from several aspects of zebrafish research progress has been collated and summarizedKey words:zebrafish Animal models Scientific research Development Infection Drug斑马鱼(Danio rerio)又名蓝条鱼、花条鱼、蓝斑马鱼、印度鱼、印度斑马鱼,产于孟加拉、印度东部、巴基斯坦、缅甸、尼泊尔等地,是一种常见的热带淡水硬骨鱼。
介绍一下斑马鱼的作文
介绍一下斑马鱼的作文
朋友们！今天咱们来聊聊一种特别有趣的小鱼——斑马鱼。
斑马鱼，光听名字就知道，它们身上有着像斑马一样的条纹。
这些条纹可
漂亮啦，黑的白的相间，整整齐齐，就像是大自然给它们精心设计的时尚装扮。
斑马鱼个头不大，小小的身体十分灵活。
它们在水里游来游去，那速度，
就像一道道闪电，嗖的一下就从这边窜到那边去了。
别看它们小，精力可旺盛
着呢！
这种小鱼性格还挺活泼，总是一群群地聚在一起，热热闹闹的，好像在开
派对。
而且它们不怎么挑食，给啥吃啥，特别好养活。
在科学研究中，斑马鱼也是大功臣呢！因为它们繁殖快，胚胎透明，科学
家们能很方便地观察它们的生长过程，研究好多生命的奥秘。
总的来说，斑马鱼不仅长得好看，还为科学做出了贡献，真是小鱼界的明
星呀！怎么样，你是不是也觉得斑马鱼很有意思呢？。
关于斑马鱼的作文
《关于斑马鱼》
我家有几条斑马鱼，它们可漂亮啦！
斑马鱼的身体细细长长的，身上有一道道蓝色和银色的条纹，就像斑马的斑纹一样，所以叫斑马鱼。
它们在鱼缸里游来游去，速度特别快。
有时候它们会一群群地聚在一起，好像在开会。
我每天都会给它们喂食。
当我把鱼食撒进去的时候，它们就会迅速地游过来，抢着吃。
有一条斑马鱼特别调皮，总是跳出水面，好像在跟我打招呼。
我喜欢看着它们自由自在地游着。
《关于斑马鱼》
我养了一些斑马鱼，它们可有趣了！
斑马鱼的颜色很鲜艳，蓝色和银色相间，特别好看。
它们的尾巴像一把小扇子，游起来一摆一摆的。
鱼缸就是它们的家。
它们有时候会躲在水草后面，好像在玩捉迷藏。
有一次，我发现有两条斑马鱼在互相追逐，我猜它们是在玩耍呢。
每天看着它们，我觉得很开心。
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》范文
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》篇一一、引言近年来，BUVSs（一种新型化学物质）在环境中的广泛存在引起了科学家的关注。
由于其在生物体内的代谢过程及其对生物体的潜在毒性效应尚不明确，因此对其在斑马鱼体内的代谢特征及毒性效应的研究显得尤为重要。
本文旨在探讨BUVSs在斑马鱼体内的代谢特征，并分析其可能产生的毒性效应。
二、BUVSs在斑马鱼体内的代谢特征1. 代谢途径与产物BUVSs进入斑马鱼体内后，主要经过肝脏进行代谢。
通过实验研究，我们发现BUVSs在斑马鱼体内主要通过羟基化、甲基化等反应进行代谢。
代谢产物主要包括羟基化BUVSs、甲基化BUVSs等。
2. 代谢速率与影响因素BUVSs在斑马鱼体内的代谢速率受多种因素影响，如个体差异、环境因素等。
实验结果显示，不同年龄、性别和健康状况的斑马鱼对BUVSs的代谢速率存在差异。
此外，环境中的其他化学物质也可能影响BUVSs的代谢过程。
三、BUVSs对斑马鱼的毒性效应1. 对生长与发育的影响实验发现，BUVSs对斑马鱼的生长与发育具有显著影响。
高浓度的BUVSs会导致斑马鱼生长迟缓，体型变小，同时还会影响其生殖系统的发育。
2. 对生理功能的影响BUVSs对斑马鱼的生理功能也具有明显影响。
实验观察到，BUVSs可引起斑马鱼体内酶活性改变，如肝酶、肾酶等，从而影响机体的新陈代谢和解毒功能。
此外，BUVSs还可能对斑马鱼的神经系统和免疫系统造成影响，导致行为异常和免疫力下降。
3. 对基因表达的影响通过基因表达谱分析，我们发现BUVSs可以影响斑马鱼体内基因的表达。
长期暴露于BUVSs环境中可能导致基因突变和染色体损伤，从而对生物体产生长期毒性效应。
四、结论与展望本研究表明，BUVSs在斑马鱼体内的代谢特征及其对生物体的毒性效应不容忽视。
通过实验研究，我们了解了BUVSs在斑马鱼体内的代谢途径、代谢速率及影响因素，并分析了其对生长与发育、生理功能及基因表达的影响。
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》范文
《BUVSs在斑马鱼体内的代谢特征及其毒性效应》篇一一、引言近年来，BUVSs（一种新型化学物质）在环境中的广泛存在引起了人们的关注。
由于其在生物体内的潜在影响，研究其代谢特征及毒性效应对于保护生态环境和人类健康具有重要意义。
本文以斑马鱼为研究对象，探讨BUVSs在斑马鱼体内的代谢特征及其毒性效应。
二、材料与方法1. 材料实验所用的BUVSs购自商业供应商，斑马鱼购自专业养殖场。
实验过程中使用的其他试剂均为分析纯。
2. 方法（1）代谢特征研究：将BUVSs暴露于斑马鱼体内，通过高效液相色谱法（HPLC）和质谱法（MS）等方法，分析BUVSs在斑马鱼体内的代谢产物及代谢途径。
（2）毒性效应研究：通过观察斑马鱼的生长情况、行为变化以及生化指标等，评估BUVSs对斑马鱼的毒性效应。
三、BUVSs在斑马鱼体内的代谢特征1. 代谢产物分析通过HPLC和MS等方法，我们发现BUVSs在斑马鱼体内发生了多种代谢反应，生成了多种代谢产物。
其中，主要的代谢途径包括氧化、还原、水解和结合反应等。
2. 代谢途径分析BUVSs在斑马鱼体内的代谢途径主要包括肝细胞内酶的催化作用和生物转化过程。
其中，肝细胞内酶的催化作用是BUVSs代谢的主要途径，生成了多种代谢中间体和最终产物。
生物转化过程则涉及了结合反应，如与葡萄糖醛酸、硫酸等结合，形成极性较强的代谢物，有利于排出体外。
四、BUVSs对斑马鱼的毒性效应1. 生长情况实验结果显示，暴露于BUVSs的斑马鱼生长速度明显减慢，体型变小。
这表明BUVSs对斑马鱼的生长发育具有一定的抑制作用。
2. 行为变化BUVSs暴露导致斑马鱼出现行为异常，如活动减少、游泳速度减慢等。
这可能是由于BUVSs干扰了斑马鱼的神经系统功能。
3. 生化指标通过检测血液中的生化指标，我们发现BUVSs暴露导致斑马鱼体内酶活性发生变化，如肝酶、肾酶等。
这表明BUVSs可能对斑马鱼的肝脏和肾脏等器官造成损伤。
介绍斑马鱼作文
介绍斑马鱼作文
《可爱的斑马鱼》
嘿，大家知道吗，有一种特别有趣的小动物叫斑马鱼。
我第一次见到斑马鱼是在朋友家。
那时候我去他家玩，就瞅见他那个大大的鱼缸里游着一群小小的、色彩斑斓的家伙。
朋友说那就是斑马鱼。
哇，我当时就被吸引住啦！这些斑马鱼啊，它们的身体就像穿着条纹衣服似的，一条一条的黑白相间的条纹可显眼了。
它们在水里游来游去，那尾巴摇来摇去的，就好像在跳舞一样，太有意思了！
我站在鱼缸前看了好久好久，就看着它们一会儿成群结队地快速游过，一会儿又慢悠悠地在那晃荡。
有一条斑马鱼特别逗，它老是自己游到一个角落里，然后转着圈圈，像是在和自己玩躲猫猫呢。
还有一次，两条斑马鱼好像在比赛谁游得快，你追我赶的，那场面可热闹啦！对了，它们吃东西的时候也特别好玩，食物一丢进去，它们就马上围过去，嘴巴一张一合的，拼命抢着吃，生怕自己少吃了一点。
这些斑马鱼真的让我觉得超级有趣，它们给我带来了好多欢乐。
现在每次想到它们，我心里都暖暖的。
真希望它们能在朋友家快快乐乐地生活下去
呀，一直做那群活泼可爱的小精灵！这就是我眼中的斑马鱼啦，是不是很特别呀！。
斑马鱼养殖市场分析报告
斑马鱼养殖市场分析报告1.引言1.1 概述概述:斑马鱼是一种颇受欢迎的观赏鱼种，由于其独特的外观和活泼的性格，越来越多的人选择将其养在家中或办公场所。
随着人们对休闲娱乐的需求增加，斑马鱼养殖市场逐渐兴起并呈现出蓬勃发展的态势。
本报告将对斑马鱼养殖市场进行深入分析，为相关行业提供参考和借鉴。
1.2 文章结构文章结构部分的内容：本报告将分为引言、正文和结论三部分。
在引言部分，将概述斑马鱼养殖市场的现状，介绍文章结构并说明报告的目的。
在正文部分，将针对斑马鱼养殖市场的现状、趋势分析以及竞争情况进行详细分析。
最后，在结论部分将对斑马鱼养殖市场的发展前景进行展望，并提出相应的市场策略建议，最终对报告进行总结。
通过这样的文章结构，将全面深入地分析斑马鱼养殖市场的情况，为读者提供全面的市场分析报告。
1.3 目的目的：本报告旨在对斑马鱼养殖市场进行全面分析，包括现状、趋势、竞争情况、发展前景和策略建议，以帮助市场参与者更好地了解市场情况，制定合理的经营策略，提高市场竞争力，促进市场健康发展。
同时，通过本报告的撰写，也可以为相关领域的研究提供一定的参考依据。
1.4 总结总结部分:通过对斑马鱼养殖市场现状、市场趋势分析和市场竞争情况的分析，可以得出以下结论：斑马鱼养殖市场整体呈现出稳步增长的态势，市场需求不断增加。
同时，市场竞争激烈，需要针对竞争对手的优势和劣势制定有效的市场策略。
在未来，斑马鱼养殖市场发展前景广阔，但也存在一定的风险和挑战。
因此，合理制定市场策略和灵活应对市场变化是至关重要的。
希望本报告对斑马鱼养殖市场的相关行业从业者和投资者有所帮助。
2.正文2.1 斑马鱼养殖市场现状斑马鱼养殖市场近年来呈现出不断增长的趋势。
随着人们对斑马鱼的需求不断增加，养殖规模也在逐渐扩大。
目前，斑马鱼被广泛用于科研实验、教学展示及观赏等领域，市场需求持续增长。
同时，由于斑马鱼生长周期短、繁殖力强，养殖成本低，因此养殖行业更加受到青睐。
《BUVSs对斑马鱼仔鱼运动行为及神经炎症通路的影响》范文
《BUVSs对斑马鱼仔鱼运动行为及神经炎症通路的影响》篇一摘要：本文通过实验探究了BUVSs（某种生物活性物质或化学物质）对斑马鱼仔鱼的运动行为以及神经炎症通路的影响。
通过对仔鱼行为模式的观察，结合相关生理学、药理学以及分子生物学方法，对BUVSs的生物学作用进行了深入分析。
实验结果表明，BUVSs在适当浓度下对斑马鱼仔鱼的运动行为有显著影响，同时揭示了其对神经炎症通路的调控作用。
一、引言近年来，关于水生生物尤其是斑马鱼的研究日益受到科学界的关注。
作为一种常用的实验动物模型，斑马鱼在生物学、药理学和神经科学等多个领域具有重要价值。
本文关注的BUVSs是一种尚未完全了解其生物活性的物质，其潜在的药理作用和生理效应尚待研究。
因此，本文旨在探究BUVSs对斑马鱼仔鱼运动行为及神经炎症通路的影响。
二、材料与方法1. 实验材料：斑马鱼仔鱼、BUVSs溶液。
2. 实验方法：（1）将斑马鱼仔鱼分为对照组和实验组，实验组给予不同浓度的BUVSs溶液处理。
（2）通过行为学观察记录法，记录并分析BUVSs处理后仔鱼的运动行为变化。
（3）运用实时定量PCR、蛋白质印迹等方法检测神经炎症通路的基因表达和蛋白质水平变化。
（4）通过组织学切片观察和神经元形态学分析，评估BUVSs对斑马鱼仔鱼神经系统的直接或间接影响。
三、结果与讨论1. 运动行为影响：通过观察记录发现，低浓度的BUVSs能够显著增加斑马鱼仔鱼的活跃度，提高游动速度和活动频率；而高浓度的BUVSs则表现出一定的抑制作用，降低仔鱼的活跃度。
这表明BUVSs在浓度方面对仔鱼的运动行为有双重效应。
该现象可能与BUVSs作用于神经递质或突触相关的生物学机制有关，但具体机制还需进一步研究。
2. 神经炎症通路影响：实验结果显示，BUVSs处理后，斑马鱼仔鱼的神经炎症相关基因表达水平发生显著变化。
在低浓度BUVSs处理下，部分抗炎基因表达上调，而促炎基因表达相对抑制；高浓度处理则表现出相反的效应。
回顾：斑马鱼的养殖
斑马鱼的养殖综述Christian Lawrence *摘要：斑马鱼成为著名的脊椎动物模式研究生物，它有很多与被作为人类疾病和发展研究模型一样深受欢迎的显著地特征：繁殖迅速，体型小，世代时间短，发育早期胚胎透明可见，同时，在其他学科上，包括动物行为学，鱼类生理学，水生生物毒理学，也深受研究者的喜爱。
尽管如此，但斑马鱼科学严谨的养殖技术却发展落后。
虽然有很多来自不同的资源的直接和间接关于斑马鱼养殖的文献信息，但几乎没有用于制定标准文案。
本篇回顾旨在使与斑马鱼的生物学特性和养殖相关的科学信息更加完整，以便提高这个重要的模式生物在科学研究中的效率，以及强调在本领域需要个更长远的研究。
关键词：斑马鱼；斑马鱼类；养殖；水产业；管理.目录1. 前言 (2)2. 斑马鱼的生活史 (3)2.1 分布与生境偏好 (3)2.2 繁殖行为 (3)2.3 生命周期 (5)2.4 食性 (5)3. 斑马鱼的养殖 (5)3.1 水的化学特性 (5)3.1.1 温度 (6)3.1.2 PH (6)3.1.3 硬度 (7)3.1.4 盐度 (7)3.1.5 DO (8)3.1.6 含氮废物 (8)4. 营养，饵料和投喂实践 (8)4.1 营养需求 (8)4.2 饵料 (9)4.3 投喂 (11)5. 繁殖及繁殖技术 (11)5.1 繁殖 (11)5.2 繁殖技术 (12)5.3 产卵率 (13)6. 幼鱼的培育 (13)6.1 幼鱼的生物学特性 (13)6.2 饵料和营养 (13)6.3 水质 (14)6.4 生长率和存活率 (15)7. 成鱼的管理 (15)7.1 饲养密度 (15)7.2 基因育种工程 (16)8.结论 (17)致谢 (17)参考文献 (17)1.前言在过去的20年来，斑马鱼在基因研究与发展（Fishman,2001）,以及最近的人类疾病和治疗药物的筛选方面（Penberthy et al.,2002;Sumanasa and Lin,2004），成为著名的脊椎动物模式研究生物。
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classified based on source, nature and mechanism of action into endogenous (metabolic and enzymatic antioxidants) and exogenous (nutrient antioxidants) or direct antioxidants,indirect antioxidants and bifunctional antioxidants.15 Direct antioxidants are redox active and short lived and they are consumed during the process and need to be regenerated to offer further protection, whereas indirect antioxidants may or may not be redox active and exhibit their antioxidant effects through up-regulation of various cytoprotective compounds and proteins such as NAD(P)H, NAD(P)H:quinone oxidoreductase 1 (NQO1), superoxide dismutase (SOD), glutathione S-transferase (GST), glutathione peroxidase (GPx), heme oxygenase-1 (HO-1), glutamate-cysteine ligase (GCL), catalase and thioredoxin.16,17Intriguingly, these cytoprotective proteins are referred as the “ultimate antioxidants,” as they have relatively long half-lives, are not consumed during their antioxidant actions, can catalyze a wide variety of chemical detoxification reactions, and are involved inregeneration of some direct antioxidants.15 There are three main cellular components involved in the regulation of antioxidant response; they are Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-related factor 2 (Nrf2), and antioxidant response elements (ARE). The Keap1-Nrf2-ARE is a major signaling pathway that regulates the battery of cytoprotective proteins at transcriptional level.13,18–22 In addition to theinduction of cytoprotective proteins, Keap1-Nrf2-ARE has multiple activation pathways for maintaining the cellular redox balance and metabolism.23–25 In short, The Keap1-Nrf2-ARE signaling pathway induces an adaptive response for oxidative stress which can otherwiselead to many inflammatory diseases including cancer, Alzheimer’s and Parkinson’s diseases,and diabetes.26–29 Thus, targeting the Keap1-Nrf2-ARE signaling pathway is being considered as a rational strategy to discover preventive and therapeutic agents referred to as antioxidant inflammation modulators (AIMs) for diseases and conditions involvingoxidative stress and inflammation.30–37 Some of Nrf2-ARE inducing agents are already in clinical trials as chemopreventive agents for cancer or as therapeutic agents for conditions involving inflammation. For example, bardoxolone methyl, a potent inducer of the Nrf2pathway, is currently under phase 3 clinical trials as an orally active, first-in-class AIM for the treatment of advanced chronic kidney disease (CKD) in patients with type 2 diabetes mellitus.38–43 While several reviews have published recently on Keap1-Nrf2-ARE pathway with emphasis on its biological functions,22,29,44–51 this review mainly focuses on thechemistry of currently known small molecule modulators of Keap1-Nrf2-ARE pathway and the high throughput screening strategies being devised to discover direct reversiblemodulators of Keap1-Nrf2 interaction as potential preventive and therapeutic agents for diseases and conditions involving oxidative stress and inflammation.2. KEAP1-NRF2-ARE PATHWAYA. Component structures and functionsKeap1-Nrf2-ARE pathway is an integrated redox sensitive signaling system which regulates from 1% to 10% of our genes. 49,52 Keap1 constitutively targets Nrf2 for ubiquitin-dependent proteasomal degradation under basal (reducing) conditions of cell growth.53,54Following exposure of cells to electrophiles or oxidative stress, Nrf2 is able to escapeKeap1-mediated degradation, translocate to the nucleus, and activate ARE-dependent gene expression of a series of antioxidative and cytoprotective proteins that include HO-1, NQO1,GCL, GPx, and several members of the glutathione S-transferase family.22,55,56 These proteins include phase II detoxification enzymes and regulatory and structural proteinswhich are essential for the metabolism, detoxification of xenobiotics, redox homeostasis and cell survival.37,45,57–59 Thereby, Keap1-Nrf2-ARE signaling system reduces the intensity of acute inflammation and induces perseverance to prevent the transformation of acute pathological conditions into chronic diseases.47,60–62NIH-PA Author Manuscript NIH-PA Author ManuscriptNIH-PA Author Manuscript1. Kelch-like ECH-associated protein 1 (Keap1)—Keap1 is a 69-kDa protein that shares some homology with actin-binding Kelch protein and serves as a negative regulator of Nrf2. The human Keap1 protein sequence contains 627 amino acid residues organized into five domains as shown in Figure 1: i) the N -terminal region (NTR), ii) the Broad complex, Tramtrack, and Bric-a-Brac (BTB), iii) the linker intervening region (IVR), iv) the Kelch domain, and v) the C -terminal region (CTR). BTB domain that attached to actin-binding proteins is responsible for homodimerization and interaction with Cullin (Cul3)-based ubiquitin E3 ligase complex for Nrf2 ubiquitination. IVR containing cysteine residues are sensitive to oxidation and nuclear export signal (NES) motif. Kelch domain has six kelch repeats (KR1-KR6) and possessing multiple protein contact sites that mediate association of Keap1 with Nrf2 (the Kelch domain interacts with Neh2 domain of Nrf2) and cytoskeleton proteins actin and/or myosin.Human Keap1 contains a total of 27 cysteine residues and seven of them (i.e., Cys 151,Cys 257, Cys 273, Cys 288, Cys 297, Cys 434, and Cys 613) are highly reactive towards ROS and electrophiles and are believed to be involved in redox sensing.50,58,63–682. Nuclear factor erythroid 2-related factor 2 (Nrf2)—Nrf2 is a bZip (basic leucine Zipper) transcription factor and a member of CNC (cap “n” collar) family of transcription factors. Human Nrf2 has 605 amino acid residues that form six conserved domains Neh1 to Neh6 (Nrf2-ECH homology; ECH-chicken analog of Nrf2) as shown in Figure 2. Neh1 has a bZip motif responsible for heterodimerization with small Maf (musculoaponeurotic fibrosarcoma) protein, and Nrf2-Maf heterodimer then binds to ARE activating geneexpression. The Neh2 domain at the N -terminus contains DLG and ETGE motifs that bind to Keap1 Kelch domain, which negatively regulates the transcriptional activity of Nrf2.Domains Neh3 at the C -terminus, Neh4, and Neh5 mediate Nrf2 transactivational activity by binding to histone acetyltransferases. Neh6 has a function of Keap1-independent negative regulation of Nrf2.69 The Nrf2 protein also contains nuclear import/localization signals (NLSs) and nuclear export signals (NESs) which regulate Nrf2 shuttling in and out of the nucleus. Human Nrf2 contain six cysteine residues, and two of the cysteines (Cys 183 and Cys 506) and two other key amino acid resiudes (Ser 40 and Tyr 568) may also regulate Nrf2localization and transactivation of target genes through oxidation and phosphorylation,respectively. Nrf2 is truly a master regulator of the antioxidant response.19,58,69–773. Antioxidant response element (ARE)—ARE, which is also known as electrophile response element or EpRE, is the cis -regulatory element containing specific DNA sequences that are present in the upstream regulatory regions of genes encoding the detoxifying enzymes and cytoprotective proteins. The consensus sequence of ARE cannot berepresented as a single sequence as AREs required for some genes are distinctly different for others.78 Nevertheless, the typical length of functionally active ARE is the 16 nucleotide sequence of 5′-T A /C Ann A /G TGA C /G nnnGC A /G -3′, where n is the variable that indicates any nucleotide. Under oxidative stress conditions, stabilized Nrf2 translocates to the nucleus,forms a heterodimer with Maf, and activates ARE-dependent gene expression. Bach1 (BTB and CNC homology-1) is another negative regulator of certain ARE-dependent genes; it associates with ARE and forms a dimer with Maf protein, preventing Nrf2 from binding to DNA under normal physiological conditions.74,78–83B. Mechanisms and regulation of ARE-dependent gene expressionKeap1 binding to Nrf2 prevents Nrf2 from entering the nucleus; it acts as a chemical sensor that controls the steady state level of Nrf2 based on cellular redox conditions.84 Thus, Keap1is also known as inhibitor of Nrf2 (INrf2). Under basal conditions, Nrf2 protein is rapidly degraded with a t 1/2 of < 20 min; Nrf2 degradation is also mediated by Keap1 via specific NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscriptubiquitin-26S proteasomal pathway.85,86 As shown in Figure 3, Keap1 forms a homodimer via BTB domain and binds to Nrf2 with its Kelch domains. Neh2 domain of Nrf2 contains two binding motifs ETGE and DLG that bind to the Kelch domains of Keap1 with different affinities (two-site substrate recognition).87 Keap1 also associates with Cul3 and brings Nrf2in close proximity to Cul3-based E3 ligase complex which mediates polyubiquitination of the lysine-rich α-helix between ETGE and DLG, and subsequent degradation of Nrf2.Under induced conditions, this Keap1-Nrf2-Cul3 assembly is disturbed and consequently Nrf2 can bypass the “Keap1 gate” and stabilized with an extended t 1/2 of up to 200min.54,86,88 Presently, two mechanistic models have been proposed for the Nrf2stabilization, namely the “hinge and latch” model and the Keap1-Cul3 dissociation model.22,89In the “hinge and latch” model, the high affinity binding of ETGE motif functions as a “hinge” to fix Nrf2 to Keap1 and the low affinity binding of DLG motif functions as a “latch” to lock down the Neh2 domain which facilitates the ubiquitination and constant degradation of Nrf2 in proteosomes.87,89,90 Upon exposure to oxidative stress, cysteine residues of Keap1 in its BTB and IVR domains are covalently modified, leading to conformational changes to the Keap1 dimer.54,76 This modified conformation of Keap1could disrupt the low-affinity DLG motif interaction, but not ETGE motif interaction (~100-fold tighter than DLG motif interaction). Therefore, the DLG motif dissociates from Keap1(latch released), whereas the ETGE motif remains associated with Keap1 (hinge attached).91Under these circumstances, the orientation of Nrf2 is not suitable for ubiquitin ligase activity by Keap1-Cul3 complex.90 As a result, Nrf2 escapes from proteosomal degradation and accumulates in the cell along with newly de novo synthesized Nrf2, translocates to the nucleus, heterodimerizes with small Mafs, and binds to ARE, leading to transcription of ARE-dependent genes.50,54Dissociation of Keap1 and Cul3 is another model proposed for Nrf2 stabilization.22,29 Underinduced conditions, covalent modification of cysteine residue(s) in Cul3 binding BTB domain of Keap1 leading to a steric clash between Keap1 and Cul3.77 This modification does not change the conformation of Keap1 but rather disrupt Keap-Cul3 E3 ligase activity via the dissociation of Keap1-Cul3 interaction.92 Other alternative mechanisms have also been proposed for Nrf2 stabilization in response to inducers, such as nucleocytoplasmic shuttling of Keap1, ubiquitination of Keap1, and Nrf2 as a direct sensor.22When redox balance of cell is restored, Nrf2 is transported back into cytoplasm anddegraded through Keap1-Cul3-mediated ubiquitination.93 Alternatively, Nrf2 depletion is promoted by the feedback mechanism of increased expression of ARE-dependent genes participating in Nrf2 degradation and negative regulation including Bach1, Keap1, and Cul3.49,94,95 The involvement of several protein kinases, including PKC, CDK, GSK and Fyn, has been implicated in the regulation of Keap-Nrf2-ARE pathway.49 In addition,complex cross talks between the Keap1-Nrf2-ARE pathway and other pathways have also been demonstrated, which are beyond the scope of this review.96–98As explained from the above mechanistic models, the process of covalent (oxidative/electrophilic) modification of sulfhydryl groups of cysteine residues in Keap1 is critical for Nrf2 regulation.54 Keap1 employs multiple sensing systems using different cysteine residues for different kinds of stimuli including electrophiles, ROS and heavy metals.50,99,100Highly reactive cysteines were consistently found in the IVR region of Keap1 and two reactive cysteine residues in this region, Cys 273 and Cys 288 are essential for Nrf2degradation. Within BTB domain, a cysteine residue, Cys151 is critical for inducible modifications of Keap1 function. Modification of these cysteine residues inhibitsNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptubiquitination of Nrf2 and stabilizes Nrf2 even in the absence of stimuli (underphysiological conditions).63,67,101,102 Recently, a cysteine residue of Nrf2 (Cys 506) was found to be involved in the direct inducer sensing function and has been suggested as a critical residue in Keap1-Nrf2 interaction.102 Clearly, Keap1 is a key component in theregulation of Nrf2, playing major roles such as an anchor for Nrf2 protein in the cytoplasm,an adaptor for Cul3-based ubiquitin E3 ligase complex for Nrf2 ubiquitination and a sensor for stress stimuli for induction of ARE-dependent genes.C. Diseases and Conditions Involving Keap1-Nrf2-ARE pathway The processes of inflammation and oxidative stress are important in the pathogenesis of many diseases. Inflammation produces large amounts of ROS and RNS that can induce oxidative damage to DNA and other cellular components; our body has evolved defensive mechanisms to protect our cells in addition to repair the DNA damage.103 The Keap1-Nrf2-ARE pathway is a common defense mechanism to counteract oxidative stress and protects multiple organs and cells.103 The protective role of Nrf2 activation has also been established in many human disorders including cancer, Alzheimer’s and Parkinson’s diseases, chronic obstructive pulmonary disease (COPD), asthma, atherosclerosis, diabetes, inflammatory bowel disease, multiple sclerosis, osteoarthritis and rheumatoid arthritis. Regulation of Nrf2-ARE signaling has also been implicated in the determination of health span, longevity, and aging.104 As discussed below, the emerging role of Keap1-Nrf2-ARE pathway in oxidative stress-related pathologies offers novel therapeutic opportunities. Pharmacological interventions are being actively pursued for the discovery of modulators of this pathway as potential preventive and therapeutic agents. This section briefly summarizes the diseases and conditions that involve Keap1-Nrf2-ARE pathway and those that could potentially be prevented or treated with modulators of this pathway.1. Cancer—The cancer chemopreventive properties have been the most intensively studied and suggested of most synthetic and natural Nrf2 inducers. Activation of Nrf2 upregulatesvarious conjugating enzymes for the detoxication of chemical carcinogens and confers protection against carcinogenicity, mutagenicity and other forms of toxicity.36,105Disruption of Nrf2 induction has increased the susceptibility of cells towards carcinogens and contributes to the inflammation progression, ultimately cancer formation.47 Cancer chemopreventive activities of Nrf2 activators have been demonstrated in several cancer models including colon cancer, bladder cancer, lung cancer, stomach cancer, breast cancer,skin cancer, and liver cancer.27 Indeed, several activators of Keap1-Nrf2-ARE pathway have been tested in clinic trials for the prevention of a variety of cancers.43,106–108Nrf2 has also been shown to be over-expressed in some cancer cells, indicating a potential role in tumor cell growth and survival.48,62,109 This dual action of Nrf2 has been recognized as a “double-edged sword” with regard to the benefits or risks of Keap1-Nrf2 system in cells.33,84,110,111 Somatic mutations or single nucleotide polymorphism in Nrf2 and Keap1genes resulted in constitutive activation of Nrf2 and promotion of tumor growth.112,113 It has been hypothesized that the cytoprotective activity of Nrf2 can be exploited by cancer cells not only to cope with the challenging tumor microenvironment, but also confer chemo-and/or radio resistance during anticancer therapies.84 Recently, it has been shown that suppression of Nrf2 activity inhibits tumor growth and enhances the efficacy of cancerchemotherapeutic agents.114,115 Thus, Nrf2 activity could be targeted for cancer treatment as well as chemoprevention though in different patient populations.2. Chronic obstructive pulmonary disease (COPD)and other airway disorders —Oxidative stress has been implicated in the pathogenesis of various airway disorders including inflammation, cell proliferation, airway obstruction, and respiratory failure.117NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptNrf2 is predominantly expressed in epithelium and alveolar macrophages and protects the lungs from oxidative stress, alveolar cell apoptosis, extracellular matrix proteolysis and chronic inflammation. The decline in Nrf2-dependent antioxidants and glutathione levels with significant decrease in Nrf2 expression was observed in COPD patient lungs.116Activation of Nrf2 is a novel therapeutic approach for the antioxidant therapy not only in COPD but also in other airway disorders associated with oxidative stress including idiopathic pulmonary fibrosis (IPF), asthma, cystic fibrosis, bronchopulmonary dysplasia (BPD), acute lung injury (ALI)/acute respiratory distress syndrome (ARDS).117–1203. Alzheimer’s and Parkinson’s diseases—Mitochondrial function is essential for neuronal signaling, plasticity, and transmitter release. Brain is sensitive to oxidative stress due to its high lipid content and oxygen consumption, and mitochondrial dysfunction and subsequent production of ROS are implicated in the pathogenesis of a wide variety of chronic neurodegenerative diseases including Alzheimer’s (AD) and Parkinson’s (PD)diseases.121 The protective effect of Nrf2 against oxidative stress and neurotoxicity has been demonstrated and Keap1-Nrf2-ARE Pathway has been proposed as a therapeutic target in AD and PD disease states.122–124 Several studies indicate that increasing nuclear Nrf2function can be protective to NT2N and dopaminergic neurons.122,125–127 Activation of Nrf2 can also potentially treat other neurological disorders such as Huntington’s disease,amyotrophic lateral sclerosis (Lou Gehrig’s disease), Friedreich’s ataxia, Down syndrome,multiple sclerosis, traumatic brain injury, and cerebral hemorrhages.30,128,1294. Atherosclerosisand heart diseases—Nrf2 is ubiquitously expressed in the cardiovascular system. Keap1-Nrf2-ARE signaling pathway is critically involved in the regulation of vascular homeostasis and also in the protection of heart against pathological cardiac hypertrophy and heart failure via the suppression of oxidative stress.130 Nrf2pathway has evolved as a therapeutic target against cardiovascular associated diseases including atherosclerosis, a leading cause for heart attack and stroke.35,131Although Nrf2was shown to have antiatherogenesis and vascular protective effects,132 it was recentlysuggested that Nrf2 promotes the atherosclerotic plaque formation via a differentmechanism.133,134 Therefore, modulation of Nrf2 may hold promise for the prevention and treatment of arteriosclerosis.5. Chronic kidney diseases (CKD)and diabetes—Nrf2-mediated expression of endogenous antioxidants has been shown as a critical adaptive defense mechanism against high glucose-induced oxidative damage in diabetes.45,110,135–137 The protective role of Nrf2in diabetic nephropathy and neuropathy has been well established, suggesting thattherapeutic activation of Nrf2 could be an effective approach to prevent as well as to treat diabetes and diabetes-related metabolic disorders.138,139 For example, CDDO-Me(Bardoxolone Methyl), a potent inducer of Nrf2 pathway, is being evaluated clinically for the treatment of CKD in patients with type 2 diabetes mellitus.386. Inflammatory bowel diseases—Increased free radicals and impaired antioxidant defenses in the intestines have been linked to the pathogenesis of inflammatory bowel diseases.140 It was suggested that Nrf2 plays an important role in protecting intestinalintegrity via regulation of pro-inflammatory cytokines and induction of phase II detoxifying enzymes.141 Therefore, Nrf2 may serve as novel target for designing therapies to prevent and treat inflammatory bowel diseases such as Crohn’s diease and ulcerative colitis.1427. Rheumatoid arthritis and Osteoarthritis—Nrf2 regulates the innate immune response and its modulation has been involved in attenuating various immune andinflammatory responses.60,143 Disruption of Nrf2 has been found to aggravate autoimmuneNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptjoint diseases such as rheumatoid arthritis and osteoarthritis.144,145 Hence, Nrf2 has been considered as a therapeutic target for arthritis and other autoimmune diseases.3. MODULATORS OF KEAP1-NRF2-ARE PATHWAY The Keap1-Nrf2-ARE pathway can be activated by various cellular stresses (including oxidative stress, endoplasmic reticulum stress, and shear stress) and chemical inducers from both endogenous (reactive oxygen and nitrogen species) and exogenous sources.27,61 A variety of small molecule inducers of ARE system have been identified from natural sources as well as from synthetic agents. Conversely, the Keap1-Nrf2-ARE is also induced by toxic chemicals that evoke oxidative stress.146 We can divide the small molecule modulators of Keap1-Nrf2-ARE pathway into ARE inducers and inhibitors based on their ultimate biological effect on ARE-mediated gene expression. While most known small molecule ARE inducers are indirect inhibitors of Keap1-Nrf2 interaction, efforts are underway to discover direct small molecule inhibitors of Keap1-Nrf2 interaction through high throughput screening or structure-based drug design approaches.A. Nrf2 Inducers Almost all currently known ARE inducers (or activators) are indirect inhibitors of Keap1-Nrf2 interaction and they are believed to form covalent adducts with the sulfhydryl groups of cysteines in Keap1 by oxidation or alkylation.43,50,100 Electrophilicity is a common property of most of the known ARE inducers. However, not all electrophiles can regulate ARE activity.86 It is well known that the biological effect of electrophiles being therapeutic to toxic depends on its hardness (‘hard’ and ‘soft’) that define the rate and selectivity of interactions with nucleophiles. Specifically, soft electrophiles will react predominantly with soft nucleophiles such as the sulfhydryl groups of cysteine, whereas hard nucleophiles target the amino and hydroxyl groups on nucleic acids and thus induce carcinogenicity and genotoxicity.48,147 Adduct formation is not only dependent on the nature of electrophile butalso on the protein microenvironment of nucleophilic center which is the function of both steric and electronic factors mediated primarily through tertiary structure of theprotein.48,148 Different classes of electrophilic inducers can display different reaction patterns with the cysteine residues of protein target in the Keap1-Nrf2 system which may lead to distinct biological effects.29,100 It has been speculated that the highly reactivecysteine sulfhydryl groups of Keap1 with low pKa values (due to proximal basic residues)render Keap1 as a unique cellular sensor for Nrf2 inducers.15,88,149–151 An alternativemechanism was proposed that switches ubiquitination of Nrf2 to Keap1 upon modification of Keap1 cysteine residues by electrophilic inducers leading to dissociation and nuclear translocation of Nrf2.152The chemistry of indirect small molecule inhibitors of Keap1-Nrf2 interaction has beenprogressively understood. Currently, known indirect inhibitors of Keap1-Nrf2 interaction are classified into about ten chemically distinct classes based on their chemical structure and their nature of reaction/interaction with cysteine sulfhydryl groups: (1) Michael acceptors,(2) oxidizable diphenols and quinones, (3) isothiocyanates and sulfoxythiocarbamates, (4)dithiolethiones and diallyl sulfides, (5) vicinal dimercaptans, (6) trivalent arsenicals, (7)selenium-based compounds, (8) polyenes, (9) hydroperoxides, and (10) heavy metals and metal complexes.49,153–155 Representative examples are discussed here in each class.1. Michael acceptors—Michael acceptors (olefins or acetylenes conjugated with electron withdrawing carbonyl groups) are a prominent class of indirect inhibitors of Keap1-Nrf2interaction. Michael acceptors are considered as soft Lewis acids and the order of ARE-inducing potency is proportional to their reactivity in the Michael addition reaction.156 As shown in Figure 4, Michael acceptors can react with the critical cysteine thiolate (soft base)NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscriptgroups in Keap1 and consequently suppress Nrf2 ubiquitination and induce the expression of ARE mediated antioxidative and cytoprotective enzymes.68,157 Michael acceptors usually show a bell-shaped dose-response curve, i.e. beneficial cellular response at low concentrations and unwanted cellular toxicity at high concentrations.158 Cytotoxicity at high concentrations is probably related to their “off-target” actions due to their chemical reactivity rather than through activation of Nrf2 signaling.48,51 Many structurally unrelated inducers are electrophilic Michael acceptors, typically found in various phytochemicals such as curcuminoids, cinnamic acid derivatives, coumarins, chalcones, flavonoids, and terpeniods.Curcumin (1), a yellow pigment from turmeric (Curcuma longa ), is a principle component of Indian curry.108 Due to the presence of two phenolic functional groups and the β-diketo moeity, curcumin is known to directly scavenge radical and other reactive oxygen species.159 Curcumin contains two Michael acceptor groups that can react readily with cysteine sulfhydryl groups. It has been shown to effectively activate Keap1-Nrf2-ARE signaling pathway presumably through modification of cysteine sulfhydryl groups in Keap1.160–162 Curcumin has also shown to indirectly activate ARE system by stimulating the upstream kinase pathways.163,164 Curcumin can not only induce the upregulation of phase II and oxidative stress response enzymes but can also inhibit carcinogen-induced expression of phase I CYP450 enzymes.165 Curcumin is highly lipophilic and rapidly metabolized by glucuronidation and sulfation conjugation reactions. Curcumin has been shown to possess a number of therapeutic properties including antiinflammatory,antioxidative, antiarthritic, antiischemic and anticarcinogenic effects and has been evaluated in clinical trials for various diseases including multiple myeloma, pancreatic cancer, colon cancer, psoriasis, and Alzheimer’s disease.107,166 A synthetic analogue of curcumin,salicylcurcuminoid (2) having ortho hydroxyl substitutions relative to the α,β-unsaturated ketone system, is much more potent (~24-fold) than curcumin in inducing quinone reductase in murine hepatoma cells.160 Bis[2-hydroxybenzylidene]acetone (3) is a another syntheticanalog of curcumin and was found to markedly increase the activities of NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1) and glutathione reductase, and the levels of totalglutathione in rapidly growing L1210 murine leukemia cells.167 The major concerns with using curcumin and analogs as preventive and therapeutic agents are their known chemical and metabolic instability, poor membrane permeability, and extremely low oralbioavailability.Cinnamic acid esters such as caffeic acid phenethyl ester (CAPE, 4) and ferulic acid ethyl ester (EFE, 5), are α,β-unsaturated esters and were shown to induce ARE system with comparable potency to curcumin.162 CAPE (4) is an active component of propolis from honeybee hives. It is known to have antimitogenic, antiinflammatory, andimmunomodulatory properties.168 Ferulic acid and its derivatives are found in many fruits and vegetables and are known to have strong protection against oxidation of protein and lipids.169,170Chalcones are open-chain flavonoids found in many plants. Chemically, their backbone consists of two phenyl rings joined by a three-carbon α,β-unsaturated carbonyl system.NIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author Manuscript。