药学学报, 2019, 54(5): 854-860
引用本文:
杨国威, 彭世泽, 赵建元, 张永欣, 岑山. 基于海分枝杆菌感染斑马鱼模型的抗结核药物评价方法研究[J]. 药学学报, 2019, 54(5): 854-860.
YANG Guo-wei, PENG Shi-ze, ZHAO Jian-yuan, ZHANG Yong-xin, CEN Shan. Application of Mycobacterium marinum infected zebrafish for evaluation of anti-tuberculosis drugs[J]. Acta Pharmaceutica Sinica, 2019, 54(5): 854-860.

基于海分枝杆菌感染斑马鱼模型的抗结核药物评价方法研究
杨国威1, 彭世泽2, 赵建元2, 张永欣2, 岑山2
1. 首都医科大学附属北京友谊医院, 北京 100050;
2. 中国医学科学院、北京协和医学院医药生物技术研究所, 北京 100050
摘要:
结核病(tuberculosis,TB)是由结核分枝杆菌导致的一种严重感染性疾病。近年来随着耐药结核菌不断出现,发展新型抗结核药物迫在眉睫。本研究利用与结核分枝杆菌高度类似的海分枝杆菌为模式生物,建立了海分枝杆菌-斑马鱼幼鱼感染模型和细菌菌数定量PCR(quantitative PCR,qPCR)分析方法。研究表明,卵黄囊注射海分枝杆菌是一种高效、便捷的感染斑马鱼胚胎方式。通过测定感染的斑马鱼存活率、斑马鱼体内细菌数目和ZiehlNeelsen抗酸性染色指标,对抗结核药物异烟肼和利福平的药效、药物之间的协同作用等多个参数进行了分析和比较,结果表明3种评价方法具有较好的一致性。本研究证实了海分枝杆菌-斑马鱼感染模型结合细菌菌数qPCR分析方法是一种简单、高效的抗结核药物体内筛选和评价系统。本研究依据中国医学科学院医药生物技术研究所《实验动物管理条例》中相关伦理规定,开展动物实验。
关键词:    海分枝杆菌      抗结核药物      斑马鱼模型      药物评价      定量PCR     
Application of Mycobacterium marinum infected zebrafish for evaluation of anti-tuberculosis drugs
YANG Guo-wei1, PENG Shi-ze2, ZHAO Jian-yuan2, ZHANG Yong-xin2, CEN Shan2
1. Beijing Friendship Hospital Affiliated to Capital Medical University, Beijing 100050, China;
2. Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China
Abstract:
Tuberculosis (TB) is a serious infectious disease caused by Mycobacterium. tuberculosis. In recent years, with the emergence of drug-resistant forms, the development of new anti-tuberculosis drugs is urgently needed. In this study, we used Mycobacterium marinum (M. marinum), which is highly similar to M. tuberculosis, to establish a M. marinum infected-zebrafish model and quantitative PCR (qPCR) method for bacterial count analysis. The results showed that injecting M. marinum into the yolk sac is an efficient and convenient way to infect zebrafish embryos. By counting the survival rate of infected zebrafish and the number of bacteria in zebrafish by ZiehlNeelsen staining, we analyzed the efficacy of isoniazid and rifampicin as anti-tuberculosis drugs and the synergistic effect of drugs. The results suggested that three evaluation methods exhibit good consistency. This study demon strated that zebrafish-M. marinum infection model combined with qPCR analysis is a simple and efficient method for in vivo screening and evaluation of anti-tuberculosis drugs. Animal experiments were carried out in accordance with the provisions for animal ethics in the Regulations on Laboratory Animals of Institute of Medicinal Biotech nology, Chinese Academy of Medical Sciences.
Key words:    Mycobacterium marinum    anti-tuberculosis drug    zebrafish model    drug evaluation    qPCR   
收稿日期: 2018-12-12
DOI: 10.16438/j.0513-4870.2018-1110
基金项目: 中国医学科学院医学与健康创新工程(2017-I2M-2-002YXZ).
通讯作者: 张永欣,Tel:86-10-63131011,E-mail:yongxinzhang@imb.pumc.edu.cn;岑山,Tel:86-10-63037279,E-mail:shancen@imb.pumc.edu.cn
Email: yongxinzhang@imb.pumc.edu.cn;shancen@imb.pumc.edu.cn
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参考文献:
[1] Jamison DT, Breman JG, Measham AR, et al. Disease Control Priorities in Developing Countries[M]. New York:The World Bank and Oxford University Press, 2006:289-309.
[2] Korenromp EL, Bierrenbach AL, Williams BG, et al. The measurement and estimation of tuberculosis mortality[J]. Int J Tuberc Lung Dis, 2009, 13:283-303.
[3] Russell DG, Barry CE 3rd, Flynn JL. Tuberculosis:what we don't know can, and does, hurt us[J]. Science, 2010, 328:852-856.
[4] Schnappinger D, Ehrt S, Voskuil MI, et al. Transcriptional adap tation of Mycobacterium tuberculosis within macrophages:in sights into the phagosomal environment[J]. J Exp Med, 2003, 198:693-704.
[5] Clay H, Volkman HE, Ramakrishnan L. Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death[J]. Immunity, 2008, 29:283-294.
[6] Kuijl C, Savage ND, Marsman M, et al. Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1[J]. Nature, 2007, 450:725-730.
[7] Ottenhoff TH. Overcoming the global crisis:"yes, we can", but also for TB...?[J]. Eur J Immunol, 2009, 39:2014-2020.
[8] Wang X, Zhu NY, Jiang W, et al. Identification of novel antituberculosis lead compound targeting shikimate kinase[J]. Acta Pharm Sin (药学学报), 2018, 53:878-886.
[9] Yang X, Zang X. Recent advances in study of mycobacterial membrane protein large 3 inhibitors[J]. Acta Pharm Sin (药学学报), 2017, 52:1379-1386.
[10] Payne DJ, Gwynn MN, Holmes DJ, et al. Drugs for bad bugs:confronting the challenges of antibacterial discovery[J]. Nat Rev Drug Discov, 2007, 6:29-40.
[11] Pethe K, Sequeira PC, Agarwalla S, et al. A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-sourcedependent growth inhibitors devoid of in vivo efficacy[J]. Nat Commun, 2010, 1:57.
[12] Mione MC, Trede NS. The zebrafish as a model for cancer[J]. Dis Model Mech, 2010, 3:517-523.
[13] Trede NS, Langenau DM, Traver D, et al. The use of zebrafish to understand immunity[J]. Immunity, 2004, 20:367-379.
[14] Amsterdam A, Hopkins N. Mutagenesis strategies in zebrafish for identifying genes involved in development and disease[J]. Trends Genet, 2006, 22:473-478.
[15] Lesley R, Ramakrishnan L. Insights into early mycobacterial pathogenesis from the zebrafish[J]. Curr Opin Microbiol, 2008, 11:277-283.
[16] Kari G, Rodeck U, Dicker AP. Zebrafish:an emerging model system for human disease and drug discovery[J]. Clin Pharmacol Ther, 2007, 82:70-80.
[17] Milan DJ, Peterson TA, Ruskin JN, et al. Drugs that induce repolarization abnormalities cause bradycardia in zebrafish[J]. Circulation, 2003, 107:1355-1358.
[18] Driever W, Solnica-Krezel L, Schier AF, et al. A genetic screen for mutations affecting embryogenesis in zebrafish[J]. Development, 1996, 123:37-46.
[19] Rudner LA, Brown KH, Dobrinski KP, et al. Shared acquired genomic changes in zebrafish and human T-ALL[J]. Oncogene, 2011, 30:4289-4296.
[20] Traver D, Herbomel P, Patton EE, et al. The zebrafish as a model organism to study development of the immune system[J]. Adv Immunol, 2003, 81:253-330.
[21] Davis JM, Ramakrishnan L. The role of the granuloma in expan sion and dissemination of early tuberculous infection[J]. Cell, 2009, 136:37-49.
[22] Martin CS, Moriyama A, Zon LI. Hematopoietic stem cells, hematopoiesis and disease:lessons from the zebrafish model[J]. Genome Med, 2011, 3:83.
[23] Tobin DM, Ramakrishnan L. Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis[J]. Cell Microbiol, 2008, 10:1027-1039.
[24] Davis JM, Clay H, Lewis JL, et al. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos[J]. Immunity, 2002, 17:693-702.
[25] Swaim LE, Connolly LE, Volkman HE, et al. Mycobacterium marinum infection of adult zebrafish causes caseating granulo matous tuberculosis and is moderated by adaptive immunity[J]. Infect Immun, 2006, 74:6108-6117.
[26] Volkman HE, Clay H, Beery D, et al. Tuberculous granuloma formation is enhanced by a Mycobacterium virulence determi nant[J]. PLoS Biol, 2004, 2:e367.
[27] Roca FJ, Ramakrishnan L. TNF dually mediates resistance and susceptibility to mycobacteria via mitochondrial reactive oxygen species[J]. Cell, 2013, 153:521-534.
[28] Tobin DM, Vary JC Jr, Ray JP, et al. The lta4h locus modulates susceptibility to mycobacterial infection in zebrafish and humans[J]. Cell, 2010, 140:717-730.
[29] Sridevi JP, Anantaraju HS, Kulkarni P, et al. Optimization and validation of Mycobacterium marinum-induced adult zebrafish model for evaluation of oral anti-tuberculosis drugs[J]. Int J Mycobacteriol, 2014, 3:259-267.
[30] Takaki K, Davis JM, Winglee K, et al. Evaluation of the patho genesis and treatment of Mycobacterium marinum infection in zebrafish[J]. Nat Protoc, 2013, 8:1114-1124.
[31] Luukinen H, Hammaren MM, Vanha-Aho LM, et al. Modeling tuberculosis in Mycobacterium marinum infected adult zebrafish[J]. J Vis Exp, 2018. DOI:10.3791/58299.
[32] Carvalho R, de Sonneville J, Stockhammer OW, et al. A highthroughput screen for tuberculosis progression[J]. PLoS One, 2011, 6:e16779.
[33] Gurtler V, Stanisich VA. New approaches to typing and identifi cation of bacteria using the 16S-23S rDNA spacer region[J]. Microbiology, 1996, 142:3-16.
[34] Roth A, Fischer M, Hamid ME, et al. Differentiation of phyloge netically related slowly growing mycobacteria based on 16S-23S rRNA gene internal transcribed spacer sequences[J]. J Clin Microbiol, 1998, 36:139-147.
[35] Clay H, Davis JM, Beery D, et al. Dichotomous role of the macrophage in early Mycobacterium marinum infection of the zebrafish[J]. Cell Host Microbe, 2007, 2:29-39.
[36] Volkman HE, Pozos TC, Zheng J, et al. Tuberculous granuloma induction via interaction of a bacterial secreted protein with host epithelium[J]. Science, 2010, 327:466-469.
[37] Adams KN, Takaki K, Connolly LE, et al. Drug tolerance in replicating mycobacteria mediated by a macrophage-induced efflux mechanism[J]. Cell, 2011, 145:39-53.