斑马鱼结核性脑膜炎模型的制备及其用于抗结核性脑膜炎药物评价的研究  

Establishing zebrafish tuberculosis meningitis models and its application in evaluating anti-tuberculous drugs

作　　者：刘茂颖 牛良飞 郭文娟 郭晓奎 宋曙 晏博 陈峥宏 LIU Maoying;NIU Liangfei;GUO Wenjuan;GUO Xiaokui;SONG Shu;YAN Bo;CHEN Zhenghong(Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University,Guiyang 550025,Guizhou,China;Shanghai Public Health Clinical Center,Shanghai 201508,China;Key Laboratory of Pathogenic Biology Characteristics of Education Department of Guizhou,Guizhou Medical University,Guiyang 550025,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550025 [2]上海市公共卫生临床中心,上海201508 [3]贵州医科大学贵州省普通高等学校病原生物学特色重点实验室,贵州贵阳550025

出　　处：《贵州医科大学学报》2025年第4期502-512,共11页Journal of Guizhou Medical University

基　　金：国家自然科学基金项目(81801977);贵州省教育厅滚动支持省属高校科研平台项目(黔教技〔2022〕019号)。

摘　　要：目的制备两种不同的斑马鱼-海分枝杆菌(Mycobacterium marinum,M.m)结核性脑膜炎模型,评价抗结核药物穿过血脑屏障清除斑马鱼脑部细菌的能力,评估模型应用于抗结脑药物评价的可行性。方法以斑马鱼幼鱼为观察对象,显微注射M.m至斑马鱼幼鱼总主静脉与后脑,制备全身性与局限性感染的结核性脑膜炎模型;通过高内涵显微镜拍摄斑马鱼体内M.m的动态变化,同时利用抗酸染色、HE染色评估两种模型的制备效果;体外测试异烟肼(isoniazid,INH)、利福平(rifampicin,RIF)对M.m菌株的最小抑菌浓度(minimal inhibitory concentration,MIC);用不同剂量的INH、RIF以总主静脉注射或浸泡给药的方式处理脑部局部感染的斑马鱼幼鱼,通过检测药物作用后斑马鱼脑部菌载量,评价药物穿过血脑屏障对脑部M.m生长的作用。结果经总主静脉注射,M.m可随血液循环生长扩散至斑马鱼幼鱼脑部,可模拟肺外(mycobacterium tuberculosis,Mtb)随血液循环系统进入脑部的感染过程;后脑注射M.m可在斑马鱼脑部引发类上皮细胞增生、炎症细胞聚集等免疫反应,并且在感染后4 d,M.m仍局限在斑马鱼脑部,此种感染方式适用于研究药物入脑的效果;INH对抑制脑部M.m生长的效果优于RIF(P<0.05),提示INH能更好的穿透血脑屏障发挥功效。结论通过总主静脉、后脑注射M.m至斑马鱼幼鱼可成功制备斑马鱼结核性脑膜炎模型,该模型可用于评估抗结核药物通过血脑屏障的能力。Objective To establish two zebrafish with Mycobacterium tuberculosis(Mtb)meningitis(TBM)models for evaluating the capacity of anti-tuberculous drugs to penetrate the blood-brain barrier(BBB)to clear bacteria and assessing the feasibility of applying the models to evaluate anti-tuberculosis drugs.Methods Zebrafish larvae were used as observation objects.Microinjection of M.marinum(M.m)into the caudal veins and the hindbrains of zebrafish larvae were establish systemic and localized TBM,respectively.M.m dynamic changes in zebrafish were captured using high-content microscopy.The preparation efficacy of two models was evaluated by Ziehl-Neelsen staining and hematoxylin and eosin(H&E)staining.Minimal inhibitory concentrations(MIC)of isoniazid(INH)and rifampicin(RIF)against M.m strain were determined using in vitro assays.Zebrafish larvae with localized brain infections were treated with different doses of INH and RIF via caudal vein injection or immersion.Determining bacterial load in zebrafish brain after drug action was used to evaluate the effect of drug penetrating BBB on brain M.m growth.Results Following caudal vein injection,M.m disseminated to the brain of zebrafish larvae via systemic circulation,mimicking the hematogenous spread of extrapulmonary Mtb to the brain.Hindbrain microinjection of M.m induced immune responses in the zebrafish brain,including epithelioid-like cell hyperplasia and inflammatory cell infiltration.Notably,M.m remained localized to the brain for at least 4 days post-infection,making this model suitable for evaluating drug penetration into the brain.INH was superior to RIF in the efficacy in suppressing intracerebral M.m growth(P<0.05),suggesting that INH had enhanced BBB penetration and exerted its effectiveness.Conclusion Caudal vein and hindbrain microinjections of M.m in zebrafish larvae successfully establish zebrafish models of tuberculous meningitis.The models are applicable for assessing the capacity of anti-tuberculosis drugs to penetrate the blood-brain barrier.

关 键 词：斑马鱼 海分枝杆菌 结核性脑膜炎模型 异烟肼 利福平 

分 类 号：R965.2[医药卫生—药理学]