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目的探讨体外培养的肥大细胞对猪布鲁菌S2株的模式识别及活化效应。方法骨髓来源的肥大细胞在体外感染猪布鲁菌S2株,瑞氏-姬姆萨染色法观察肥大细胞形态学变化,甲苯胺蓝染色法观察肥大细胞脱颗粒情况;间接ELISA检测S2株感染后1 h和12 h肥大细胞释放组胺、IFN-γ、IL-12的含量;激光共聚焦显微镜检查肥大细胞对S2株的摄取状态;RT-PCR法观察S2株感染后12 h和24 h肥大细胞TLR4和TLR8基因的mRNA的表达;流式细胞术观察S2株感染后24 h肥大细胞TLR4和TLR8蛋白的表达。结果感染猪布鲁菌S2株的肥大细胞形态出现了明显的变化,S2株感染后1 h即出现明显的脱颗粒现象;S2株感染后1 h、12 h肥大细胞上清中组胺含量明显高于正常对照组(P<0.05),未检测到IFN-γ、IL-12;共聚焦显微镜检查发现感染30 min和1 h S2株主要黏附在肥大细胞表面,而未被肥大细胞摄入到细胞内;S2株感染12 h后肥大细胞TLR4mRNA的表达明显高于正常对照组,24 h时表达又减弱,和正常对照组相比,S2株感染24 h时肥大细胞TLR4蛋白的表达增加;S2株感染12 h和24 h后肥大细胞TLR8 mRNA和蛋白的表达无变化。结论 S2株能黏附在肥大细胞的表面并能激活肥大细胞,引起其脱颗粒,释放组胺,但在实验观察的时间点内未见IFN-γ和IL-12的分泌,其机制可能是S2株通过TLR4被肥大细胞识别,但不能被肥大细胞摄取。
Objective To investigate the pattern recognition and activation of porcine Brucella S2 in vitro by mast cells cultured in vitro. Methods Bone marrow-derived mast cells were infected in vitro with S2 strain of Brucella brucei. Morphological changes of mast cells were observed by Wright-Giemsa staining, and degranulation of mast cells was observed by toluidine blue staining. After S2 infection The levels of histamine, IFN-γ and IL-12 released by mast cells at 1 h and 12 h were measured by laser confocal microscopy. The uptake of S2 by mast cells was detected by confocal laser scanning microscopy. The expression of TLR4 and TLR8 mRNA was detected by flow cytometry. The expression of TLR4 and TLR8 in mast cells 24 hours after infected with S2 was observed by flow cytometry. Results The morphological changes of mast cells in swine brucellosis S2 showed obvious changes. S2 showed obvious degranulation 1 h after infection. The histamine in supernatant of mast cells of S2 was significantly higher at 1 h and 12 h IFN-γ and IL-12 were not detected in the normal control group (P <0.05). Confocal microscopy showed that the S2 strain was mainly adhered on the surface of mast cells at 30 min and 1 h after infection, but not by mast cells The expression of TLR4 mRNA in mast cells was significantly higher than that in the normal control group after 12 h infection of S2 strain, and the expression of TLR4 protein was also decreased at 24 h in S2 group. Compared with the normal control group, the expression of TLR4 protein increased in mast cells S2 There was no change in the expression of TLR8 mRNA and protein in mast cells at 12 h and 24 h after infection. Conclusion S2 can adhere to the surface of mast cells and activate mast cells, which can cause degranulation and release of histamine. However, the secretion of IFN-γ and IL-12 was not observed at the experimental time points. The mechanism may be S2 Strains are recognized by mast cells via TLR4 but not by mast cells.