肥大细胞对布鲁菌S_2株的模式识别及活化效应的体外研究

Pattern recognition and activation effect of mast cells in vitro infected by Brucella suis S_2

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 study the pattern recognition and activation effect of mast cells infected by Brucella （B.） suis S2. Methods Mast cells dedved from bone marrow in vitro were infected by B. suis $2. The change in the cell morphology was observed with Wrights-Giemsa＇s staining, and cell degranulation was tested with toluidine blue staining. The extracellular levels of histamine, IFN-y and IL-12 of mast cells at 1 and 12 h after infection were detected by indirect ELISA. The uptake pattern of mast cells to B. suis $2 was determined by laser-scanning confocal microscopy. The expressions of TLR4 and TLR8 mRNA were detected by RT-PCR at 12 and 24 h after infection by B. suis $2, and the TLR4 and TLR8 protein expressions were detected by flow cytometry at 24 h. Results The form of mast cells infected by B. suis $2 was obviously changed. Significant degranulation was observed at ] h, and at 1, 12 h post-infection by B. suis $2, the content of histamine secreted by mast cells was significantly higher than that of normal control group （ P 〈 0.05）, and IFN-y and IL-]2 were not found. At 30 min and 1 h, B. suis $2 bound to the mast cell surface and were not uptaken into the mast cells. Compared with the control group, the expression of TLR4 mRNA increased after 12 h infection by B.suis $2, and was reduced at 24 h. The expression of TLR4 protein rose at 24 h, but the expression of TLR8 mRNA and protein did not alter at 12 and 24 h after infection by B. suis S2. Conclusion B. suis S2 can bind to the cell surface and activate mast cells, cause their degranulation, induce the release of histamine, but IFN-y and IL-12 were not found during the observing time. The mechanism may be that B. suis S2 can be recognized by mast cells through TLR4 but can not be phagocytosed by mast cells.