抗肿瘤新药研究的新型模式生物斑马鱼

斑马鱼（Danio rerio），是一种用于研究脊椎动物胚胎学和发育遗传学的模式生物。近年来，利用斑马鱼来建立人类疾病研究模型已成为新的研究亮点。笔者就斑马鱼作为一种抗肿瘤新药研究模式生物所具有的优势、应用研究及其前景做一综述。     

阿尔茨海默病斑马鱼模型的研究现状及其应用

斑马鱼作为一种新兴的脊椎模式动物,被广泛用于多种疾病的研究,包括心血管疾病、中枢神经系统疾病、肾脏疾病等。斑马鱼与人类基因高度同源,具有体外受精、体外发育、个体透明等特点,基于斑马鱼构建的疾病模型极大地推动了相关领域的基础性研究。本文综述了近年来运用斑马鱼构建的阿尔茨海默病相关模型的研究现状及其应用,为斑马鱼在阿尔茨海默病新药开发研究中的应用提供新的思路。     

人乳腺癌斑马鱼移植瘤模型建立

目的研究人乳腺癌斑马鱼移植瘤模型的建立方法及其相关的蛋白表达。方法分别选用MCF-7、T-47D、MDA-MB-231细胞系显微注射至斑马鱼幼鱼体内,考察不同乳腺癌细胞系在斑马鱼体内的定植迁移、细胞数量变化、细胞分布以及对斑马鱼肠下静脉丛(subintestinal veins,SIVs)的影响,建立乳腺癌斑马鱼移植瘤模型;并考察JAK抑制剂对斑马鱼移植肿瘤的影响,并对相关蛋白表达的情况进行蛋白印迹分析。结果在接种细胞数大于每只200时,斑马鱼的肿瘤模型成功率大于比例0.90;MB-231移植瘤在斑马鱼体内具有明显的迁移特征,这种特征可被JAK抑制剂tofacitinib抑制(P<0.01);同时tofacitinib可明显减少移植瘤模型体内MB-231的数量(P<0.01);移植瘤可促进SIVs增生,酪氨酸抑制剂PTK787可明显地抑制移植瘤引起的SIVs的生长(P<0.01);蛋白印迹结果显示,4 d斑马鱼胚胎自身HER2有表达,T-47D斑马鱼模型组组织中HER2表达增加;MB-231斑马鱼模型组组织中VEGFa高表达;tofacitinib处理后的移植瘤其p53表达增强。结论人源肿瘤通过微量显微注射方法可成功建立斑马鱼乳腺癌移植瘤模型,该模型可用于抗肿瘤药物的初步筛选研究。     

模式生物斑马鱼在心功能评价中的应用

斑马鱼繁殖力强、体积小、易饲养、发育快速、胚胎透明,与人类基因组同源性高,心脏结构和功能与哺乳类动物高度相似。基于斑马鱼模型的药物实验具有用药量少、周期短、指标易观察等优势,近年来斑马鱼模型在药物心血管安全性评价和心脏保护活性筛选领域得到了广泛应用。结合课题组前期研究及国内外文献,综述斑马鱼心功能评价应用进展,以期为斑马鱼在心血管药物安全性和活性评价中的应用提供参考。     

斑马鱼模型在药物非临床毒代动力学研究中的应用

斑马鱼是一种简单的生物实验动物,拥有和哺乳动物相似的生物结构与生理功能。斑马鱼模型在药物毒代动力学的研究应用前景十分广阔,采用斑马鱼模型研究药物在体吸收、分布和代谢过程,能够减少实验研究的假阴性或假阳性结果,提高实验准确性;采用斑马鱼模型进行一般毒理学实验的毒代动力学研究,有助于一般毒理学实验研究的剂量选择和靶器官确定;采用斑马鱼模型进行生殖与发育毒性实验的毒代动力学研究,可以提高生殖与发育毒性实验的预测性。本文主要就以上几个方面对药物在斑马鱼体内的吸收、分布和代谢过程以及该模型在药物非临床毒代动力学中的应用进行论述。     

斑马鱼炎症模型及其在中药抗炎领域的应用

中药抗炎在近年来已成为一个重要的研究领域,中药制剂的抗炎临床研究已经取得了一定进展,但具体活性成分及作用机制仍需进一步明确。斑马鱼由于其独特的生物学优势,目前已被广泛应用于中药抗炎活性的筛选。综合近年来国内外有关斑马鱼模型在中药抗炎领域的报道,从已构建的斑马鱼炎症模型、斑马鱼模型中的炎症介质、斑马鱼模型在中药抗炎药物筛选中的应用3个方面对斑马鱼炎症模型及其在中药抗炎领域的研究进展进行综述,以期为模式生物斑马鱼在中药抗炎领域的研究应用提供新思路,为抗炎中药的开发与利用提供借鉴参考。     

基于斑马鱼模型的鹿胶益气补血相关功效研究

目的 通过斑马鱼模型快速观察鹿胶补血、健骨、抗衰老等补益作用。方法 利用草甘膦(Gly)构建斑马鱼成鱼红细胞损伤模型,通过甲氨蝶呤(MTX)构建斑马鱼幼鱼造血功能损伤模型。通过泼尼松龙(Pred)构建斑马鱼幼鱼骨形成抑制模型。通过邻苯二甲酸二丁酯(DBP)构建斑马鱼幼鱼眼部细胞凋亡模型。结果 鹿胶可改善Gly诱导的斑马鱼成鱼红细胞核异常,促进造血因子SCL、GATA1的表达;改善Pred诱导的斑马鱼幼鱼骨形成抑制,促进成骨相关ALP、Runx2a的表达;减少DBP引起的斑马鱼幼鱼眼部细胞凋亡数,促进抗凋亡因子Bcl-2的表达。结论 鹿胶具有较好的保护红细胞、促进造血、促进骨形成、抗凋亡等作用,这些作用可能与其补血、抗骨质疏松、抗衰老等功效有关,该研究为鹿胶的临床应用提供科学依据,为进一步开发应用奠定基础。     

基于模式生物斑马鱼模型的中药药效物质筛选技术及应用

目的作为一个复杂成分体系,中药药效物质的精准辨识和筛选是中药现代化研究的一个瓶颈问题,探索新的实验技术十分重要。方法斑马鱼是新兴的模式生物,具有体外受精、胚胎透明,不需要解剖可方便在显微镜下观察组织器官的发育等生物学优势,可在细胞板中完成实验,需要样品量少,已被欧洲替代法验证中心推荐为新的替代动物。斑马鱼全基因组测序结果显示斑马鱼与人的基因组相似性达87%,大量的研究表明斑马鱼对药物反应与临床反应也高度相似,为斑马鱼应用于研究人类疾病与药物筛选奠定了理论和实践基础。本实验室利用斑马鱼模型构建了较为齐全的中药药效物质筛选的技术体系,并对甘草、阿胶、栀子、乳香等传统中药进行药效或毒性成分的筛选辨识研究。结果本实验室利用斑马鱼模型建立了可涵盖肿瘤、心血管、神经系统、骨骼、皮肤、重要器官等的活性/毒性筛选技术平台。从前期大规模的筛选中,发现甘草具有一定的抗肿瘤作用,进一步利用斑马鱼活性跟踪技术和高速逆流色谱分离技术,发现了2个未报道的抑制血管生成的活性成分。新阿胶是山东福胶集团的独家品种,是以猪皮为来源,作为阿胶的替代品,但是现代药理学数据缺乏导致市场认可度低,因此我们利用斑马鱼模型系统开展了新阿胶的化疗损伤后的"扶正"与"生血"的作用机制。中药栀子临床发现具有防治血栓的功效,但是抗血栓的药效标志物并不清楚,作者采用斑马鱼模型整合代谢组学技术,发现3个代表性的抗血栓成分。乳香在历代典籍中具有"孕妇慎服"记载,但是原因不明,我们利用斑马鱼模型构建发育毒性模型,并结合灰色关联度分析法,证明乳香具有一定的发育毒性,并发现了潜在的毒性靶器官,并发现了主要的毒性效应成分。结论斑马鱼作为一种新型的模式生物,在中药药效物质筛选、活性评价、毒性预警方面都具有广阔的应用前景。     

Malthus和Logistic模型及其医学应用

Malthus模型和Logistic模型是种群生态学的核心理论之一,它们在医学中的应用涉及传染病模型、肿瘤生长、肿瘤治疗等。介绍了Malthus模型和Logistic模型在医学中的主要应用。     

新型模式生物斑马鱼及其胚胎在人类疾病模型研究中的应用

斑马鱼作为一种理想的模式动物在遗传学,发育生物学的研究中应用越来越广泛。由于斑马鱼具有容易获得,易于饲养,生殖周期短,繁殖能力强等特点,且其基因组测序的完成及与人类的高度保守性,使得斑马鱼及其胚胎在关于人类疾病的研究中有着重要的应用和实际价值。本文就斑马鱼及其胚胎在人类疾病研究中的应用作一简要概述。     

Potential of zebrafish for anticancer drug screening

Background: The zebrafish is a powerful vertebrate model organism for large scale genetic and chemical screens. Objective: With the rapid development of cancer modeling in zebrafish, great opportunities exist for chemical screens to find anticancer agents. Methods: Using embryos, pathway screens can be designed based on our knowledge of cancer biology. Anticancer chemical screens using tumor bearing adult zebrafish facilitate preclinical testing. Conclusion: Current developments in improving cancer models and automated imaging technology have the potential of finding novel compounds that disrupt tumor biology.     

Human tumor xenografts and mouse models of human tumors: re-discovering the models

Background: Human tumor xenografts constitute the major efficacy and tumor biology models for cancer drug discovery. Gene expression and protein expression technologies have improved understanding of the malignant diseases represented by human tumor lines. Genetically engineered mouse (GEM) models of cancer are improving and allow assessment of immune system-mediated therapeutics. Objective: The current brief review discusses the status of human tumor xenografts, GEM models and normal tissue assays used in cancer drug discovery. Results: The Preclinical Pediatric Tumor Program consortium with 47 molecularly well-characterized pediatric tumor xenografts is leading the field. Individual laboratory studies show no correlation between culture and in vivo characteristics of some human tumor lines. The TRAMP and RIP1TAG2 GEM models have been used for efficacy studies. Recent GEM models often have two mutated genes leading to greater genetic instability. Host normal cells are important contributors in malignant disease. While convenient and frequent subcutaneous tumor implantation does not provide an optimal microenvironment for modeling clinical disease. Orthoptopic tumor implantation is gaining in use due to the availability of labeled tumor sub-lines and non-invasive imaging techniques. Conclusion: The science associated with human tumor xenografts and GEM cancer models is not static. The application of gene and protein expression technologies, the wider availability of non-invasive imaging and improved genetic engineering are allowing improved tumor modeling. However, as more is learned about current models, it is becoming clear that there is a shortage of models to cover the broad spectrum of human malignant disease.     

人源性肝癌组织移植模型研究进展

人源性肿瘤组织移植(Patient-derived human tumor xenograft,PDX)模型能很好的保持原代肿瘤的组织病理学、生长及转移特性,并在多次传代后仍然能够在较大程度上保留原代肿瘤的这些特性,与原始患者肿瘤密切相关,具有重要科研价值。肝癌PDX模型已用于患者的个性化药物筛选、药物疗效的评价、治疗方向的探索、肝癌的发生发展机制等,具有良好的应用前景。该文对肝癌PDX模型的方法、应用以及成瘤因素等方面进行综述。     

在肿瘤研究中转基因小鼠模型的研究进展

随着基因工程技术的飞速发展,转基因小鼠模型对研究肿瘤基因特征、肿瘤抑制基因的表达和功能有着重要的作用。近年来,肿瘤血管生成理论成为肿瘤研究的新热点。与肿瘤血管生成相关的调控基因的研究也越来越受到重视。该文对转基因小鼠模型在肿瘤研究中的进展作了综述,并着重介绍了关于肿瘤血管生成调控基因转基因小鼠模型的研究情况。     

Zebrafish: An emerging model system to study liver diseases and related drug discovery

The zebrafish has emerged as a powerful vertebrate model for studying liver‐associated disorders. Liver damage is a crucial problem in the process of drug development and zebrafish have proven to be an important tool for the high‐throughput screening of drugs for hepatotoxicity. Although the structure of the zebrafish liver differs to that of mammals, the fundamental physiologic processes, genetic mutations and manifestations of pathogenic responses to environmental insults exhibit much similarity. The larval transparency of the zebrafish is a great advantage for real‐time imaging in hepatic studies. The zebrafish has a broad spectrum of cytochrome P450 enzymes, which enable the biotransformation of drugs via similar pathways as mammals, including oxidation, reduction and hydrolysis reactions. In the present review, we appraise the various drugs, chemicals and toxins used to study liver toxicity in zebrafish and their similarities to the rodent models for liver‐related studies. Interestingly, the zebrafish has also been effectively used to study the pathophysiology of nonalcoholic and alcoholic fatty liver disease. The genetic models of liver disorders and their easy manipulation provide great opportunity in the area of drug development. The zebrafish has proven to be an influential model for the hepatic system due to its invertebrate‐like advantages coupled with its vertebrate biology. The present review highlights the pivotal role of zebrafish in bridging the gap between cell‐based and mammalian models.     

Zebrafish embryos as models for embryotoxic and teratological effects of chemicals

The experimental virtues of the zebrafish embryo such as small size, development outside of the mother, cheap maintenance of the adult made the zebrafish an excellent model for phenotypic genetic and more recently also chemical screens. The availability of a genome sequence and several thousand mutants and transgenic lines together with gene arrays and a broad spectrum of techniques to manipulate gene functions add further to the experimental strength of this model. Pioneering studies suggest that chemicals can have in many cases very similar toxicological and teratological effects in zebrafish embryos and humans. In certain areas such as cardiotoxicity, the zebrafish appears to outplay the traditional rodent models of toxicity testing. Several pilot projects used zebrafish embryos to identify new chemical entities with specific biological functions. In combination with the establishment of transgenic sensor lines and the further development of existing and new automated imaging systems, the zebrafish embryos could therefore be used as cost-effective and ethically acceptable animal models for drug screening as well as toxicity testing.     

GBR12909 possesses anticonvulsant activity in zebrafish and rodent models of generalized epilepsy but cardiac ion channel effects limit its clinical utility

BACKGROUND/AIMS: GBR12909 has been reported to possess anticonvulsant activity with focal brain perfusion to the hippocampus of pilocarpine, although an earlier publication suggested any anticonvulsant effects were only mild. Here we further explored the anticonvulsant potential of GBR12909 with a suite of anticonvulsant assays in both zebrafish and mammals and then explored whether it possessed any QT effects which might limit clinical utility. METHODS: We assessed the anticonvulsant effects of GBR12909 in zebrafish pentylenetetrazole (PTZ), mammalian maximal electroshock and PTZ models of generalized epilepsy and a rodent hippocampal kindling model. Cardiac effects were assessed in zebrafish and man. RESULTS: GBR12909 possesses anticonvulsant activity in zebrafish and rodent models of generalized epilepsy. However, phase 1 human data indicated potential QT effects. Subsequent testing in a zebrafish QT assay confirmed marked arrhythmogenic potential. CONCLUSION: Further clinical development of GBR12909 in epilepsy was considered inappropriate because of insufficient window between the therapeutic effects and the cardiac arrhythmia problems identified in zebrafish assays. Any further development based on this mechanism of action should avoid the GBR12909 chemical scaffold, or involve structure-activity dissociation of its neurological and cardiac effects.