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【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plc B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plc B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。
[Objective] The zebrafish living environment in many microorganisms, will activate its innate immune system, thus affecting the relevant test results, it is necessary to establish a zebrafish sterile culture system suitable for infection and immunization. 【Method】 The culture process of sterile isolator based on short-time sterilization of fertilized egg and temperature-controlled positive pressure was established. The microbiological tests of zebrafish embryos and their living environment were carried out according to the criteria of aseptic animals. Sterile zebrafish The expression of innate immunity-related genes (TLRs) was tested. Listeria monocytogenes infection test was carried out by using sterile zebrafish embryo model. 【Result】 The study successfully established a sterile operating system for zebrafish breeding. The result of sterility test showed that the living environment and the body were free of pathogenic microorganisms. The expression of TLRs in innate immune molecules was low or not expressed. However, These genes are highly transcribed in sterile zebrafish soaked infected with Listeria monocytogenes. Aseptic zebrafish are sensitive to intravenous ingestion of EGEs by L. monocytogenes. 100 CFU infection can cause all of the fish to die within 1 week, whereas their mpl or plc B deletion significantly decreases lethality ( 70% and 40% respectively). Macrophages accumulate around the intestine of infected parental strains of EGDe soaking in fish, whereas this aggregation is almost undetectable in fish infected with the mpl or plc B gene deletion strains. 【Conclusion】 Sterile zebrafish embryos obtained by simple culture system can be used in the study of innate immunity and pathogens infection.