鰤诺卡氏菌对大口黑鲈头肾巨噬细胞的侵染过程

Infection process of primary macrophages derived from kidney of Micropterus salmoides by Nocardia seriolae

摘要:

【背景】鱼类诺卡氏菌病潜伏期和病程较长，感染率和死亡率较高，给水产养殖业带来较大的经济损失，其病原鰤诺卡氏菌(Nocardia seriolae)是胞内寄生菌，侵入细胞后引起慢性感染是主要的致病机制。【目的】构建鰤诺卡氏菌侵染大口黑鲈(Micropterus salmoides)头肾巨噬细胞体外模型，观察鰤诺卡氏菌侵染巨噬细胞的过程并探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【方法】采用密度梯度离心法分离巨噬细胞，通过特异性染色和PCR扩增巨噬细胞表达基因mpeg1对细胞进行鉴定，并通过CCK-8法和氧呼吸暴发活性测定检测巨噬细胞的活性；通过倒置荧光显微镜和流式细胞术观察侵染过程中细菌与细胞的形态与数量变化；通过双荧光流式细胞术检测、乳酸脱氢酶(lactate dehydrogenase, LDH)释放试验及线粒体膜电位检测，探究鰤诺卡氏菌对巨噬细胞的凋亡作用。【结果】从大口黑鲈头肾分离获得纯度高的巨噬细胞，经染色和PCR法鉴定为巨噬细胞；筛选出最优的体外培养条件为1640培养基+1%青霉素链霉素+1%胎牛血清。在脂多糖刺激后，巨噬细胞的氧呼吸暴发能力显著提高(P<0.05)。GFP-鰤诺卡氏菌侵染细胞2 h后细菌被细胞吞噬，4 h细胞变圆且贴壁率降低，6 h细菌大量繁殖并包围细胞，8 h后细胞大量死亡。凋亡相关实验结果表明，侵染初期巨噬细胞凋亡率增加，LDH释放增加，线粒体膜电位下降；随着侵染时间延长，细胞凋亡率下降、LDH释放量及线粒体膜电位下降减少，说明鰤诺卡氏菌对巨噬细胞起先促进后抑制凋亡的作用。【结论】通过密度梯度离心法成功分离大口黑鲈头肾巨噬细胞，并通过鰤诺卡氏菌侵染细胞后初步摸清鰤诺卡细菌在细胞水平的致病机理，建立了鰤诺卡氏菌侵染大口黑鲈头肾巨噬细胞的体外模型；证实了鰤诺卡氏菌可侵染巨噬细胞并抑制细胞凋亡，从而达到在巨噬细胞内存活，为进一步开展鰤诺卡氏菌与巨噬细胞相互作用并阐明鰤诺卡氏菌的致病机制奠定了研究基础。

Abstract:

[Background] Nocardiosis has a long incubation period and long disease duration, with high infection rate and mortality rate, which brings serious economic losses to aquaculture. Nocardia seriolae, the pathogen causing Nocardiosis, is an intracellular bacterium. The main pathogenesis of this disease is the chronic infection of N. seriolae. This pathogen invading largemouth bass (Micropterus salmoides) can cause white nodules, the products of the interaction between pathogen and macrophages. The bacteria are wrapped by necrotic cells in the white nodules, which make it difficult for antibiotic to kill the bacteria, resulting in death of fish. [Objective] To establish an in vitro model for the infection of largemouth bass head kidney macrophages by N. seriolae, observe the progression of infection, and elucidate the infection-induced apoptosis of macrophages. [Methods] The macrophages were isolated by density gradient centrifugation and identified by morphological observation, specific staining, and PCR amplification of macrophage-expressed gene mpeg1. The activity and function of macrophages were detected by CCK-8 method and oxygen respiratory burst activity assay. The morphological and quantitative changes of N. seriolae and macrophages during infection were observed via inverted fluorescence microscopy and flow cytometry. The apoptosis of macrophages was investigated by double fluorescence flow cytometry, lactate dehydrogenase (LDH) release assay, and mitochondrial membrane potential assay. [Results] High-purity macrophages were isolated from head kidney of largemouth bass and were identified as macrophages by staining and PCR methods. The medium was optimized as 1640 medium + 1% penicillin-streptomycin + 1% fetal bovine serum, in which the cells could survive for 72 h in vitro, with the survival rate as high as 80%±1.03% within 24 h. The oxygen respiratory burst activity of macrophages enhanced after lipopolysaccharide stimulation (P<0.05). The GFP-N. seriolae were phagocytosed by the macrophages at 2 h, became rounded with decreased adherence rate at 4 h, and proliferated and surrounded the macrophages at 6 h. A number of macrophages died at 8 h. The initial stage of infection witnessed the increase in the apoptosis rate of macrophages, the increase in the release of LDH, and the decrease in the mitochondrial membrane potential. With the prolongation of infection, the apoptosis rate, the amount of LDH released, and the mitochondrial membrane potential decreased. It implied that apoptosis was promoted in the initial of infection and then inhibited as the infection prolonged. [Conclusion] This study successfully established an in vitro model for the infection of largemouth bass head kidney macrophages by N. seriolae. It confirmed that N. seriolae infected and survived in macrophages by inhibiting apoptosis. This study provided information for further studying the interaction between N. seriolae and macrophages and elucidating the pathogenic mechanism of N. seriolae.