斑马鱼胚胎的简易无菌培养体系及单增李斯特菌感染试验

A simplified system for generation of germ-free zebrafish embryos and its application in Listeria monocytogenes infection

摘要:

[目的] 斑马鱼的生活环境中微生物众多，会激活其先天免疫系统，从而影响相关试验结果，因此需建立适合于感染与免疫研究的斑马鱼无菌培养体系。[方法] 建立了基于受精卵短时消毒和温控正压无菌隔离器的培养流程；根据无菌动物标准对斑马鱼胚胎及其生活环境进行微生物检测；并通过定量PCR检测无菌斑马鱼先天免疫相关基因（TLRs）表达水平；利用无菌斑马鱼胚胎模型进行了单增李斯特菌感染试验。[结果] 本研究成功建立了斑马鱼培育的无菌操作系统，无菌检验结果显示其生活环境及体内不含病原微生物，先天免疫分子TLRs表达量较低或不表达，而常规培养斑马鱼以及浸泡感染单增李斯特菌的无菌斑马鱼中这些基因转录水平较高。无菌斑马鱼对单增李斯特菌强毒株EGDe静脉注射感染很敏感，100 CFU感染量能导致鱼在1周内全部死亡，而其mpl或plcB基因缺失株感染后致死率显著下降（分别为70%和40%）。巨噬细胞在亲本株EGDe浸泡感染的鱼肠道周围聚集，而mpl或plcB基因缺失株感染的鱼中几乎观察不到这一聚集现象。[结论] 通过简易培养体系获得的无菌斑马鱼胚胎可用于先天免疫和病原感染机制等研究。

Abstract:

[Objective] Under conventional cultivation conditions zebrafish harbors numerous microbes from the environment, leading to activation of its innate immune systems and interfering the results of relevant studies. We aimed to establish a germ-free zebrafish embryo model suitable for studies of host immune responses to infections.[Methods] A germ-free cultivation process including simple disinfection of the fertilized eggs and growth in a positive-pressured thermostatic isolator. Sterility testing of germ-free zebrafish embryos and water samples was done according to the national standards. The transcriptional level of TLRs, the mark genes indicating activation of the innate immune system, was detected by qPCR. Listeria monocytogenes was used as an infection model.[Results] The cultivation system and disinfection process could ensure germ-free status as shown by absence of microbes in zebrafish embryos and egg water. TLRs were barely detectable in zebrafish raised in the germ-free system, but highly induced in conventionally raised zebrafish or in germ-free zebrafish immersion-infected with pathogenic Listeria monocytogenes. The germ-free fish was sensitive to infection by L. monocytogene EGDe at a 100-CFU dose with 100% mortality in one week, while its isogenic mutants Δmpl and ΔplcB exhibited reduced death (70% and 40%, respectively). Macrophages were recruited around the intestine in EGDe immersion infected fish, but not in Δmpl and ΔplcB infected fish.[Conclusion] Zebrafish embryos produced by this simple process were free of microbes and could be used to study the innate immune responses and the pathogenesis of microbial pathogens.