金黄色葡萄球菌肠毒素B对LAD2肥大细胞系活化作用的研究

Role of Staphylococcus aureus enterotoxin B in activation of LAD2 mast cells

目的 用LAD2肥大细胞系与金黄色葡萄球菌肠毒素B（SEB）共培养，探讨SEB对肥大细胞非IgE机制的活化作用。方法 建立LAD2与不同浓度的SEB（0.01、0.1、1、10、100 μg/ml）以及A23187阳性对照、阴性对照的孵育体系，培养箱中培养30 min后，光镜下观察SEB对肥大细胞的细胞形态影响并收集各体系上清液，通过酶活性法检测肥大细胞分泌的类胰蛋白酶浓度，并通过ELISA法检测组胺水平。结果 LAD2与SEB共孵育30 min后,光镜下可见肥大细胞形态为细胞略增大、边缘模糊、突起增多，折光性下降，呈放射状毛刺样。LAD2与不同浓度SEB（0.01、0.1、1、10、100 μg/ml）共孵育30 min后，上清液检测类胰蛋白酶浓度分别为（4.116 ± 0.651）、（5.344 ± 0.874）、（3.806 ± 0.459）、（1.309 ± 0.247）、（0.310 ± 0.199） ng/ml， SEB浓度在0.01、0.1、1 μg/ml时，释放类胰蛋白酶浓度明显高于阴性对照组（P＜0.05），而SEB浓度继续增加则类胰蛋白酶浓度水平依次下降。不同SEB浓度下检测组胺水平分别为（242.409 ± 63.915）、（522.491 ± 73.466）、（550.926 ± 84.466）、（334.397 ± 33.640）、（226.527 ± 5.678） ng/ml，SEB浓度在0.01、0.1、1 μg/ml时，肥大细胞释放的组胺浓度随SEB浓度的增加而增高，明显高于阴性对照组（P＜0.05）；而SEB浓度继续增加时，肥大细胞释放组胺浓度依次下降。结论 SEB可通过非IgE依赖机制直接作用于肥大细胞，使肥大细胞活化发生形态变化并释放类胰蛋白酶和组胺。

Objective To investigate the role of Staphylococcus aureus enterotoxin B （SEB） in non-IgE mediated activation of mast cells （MCs） by in vitro co-culture of laboratory of allergic disease 2（LAD2） cells and SEB. Methods The LAD2 cells were incubated with SEB at different concentrations of 0.01, 0.1, 1, 10 and 100 μg/ml, A23187 positive control and negative control separately for 30 minutes. Then, effects of SEB on the morphology of MCs were observed by using a light microscope, and culture supernatants of the above incubation systems were collected. The concentration of tryptase released from MCs was analyzed by enzymatic activity assay, and the level of histamine was detected by enzyme-linked immunosorbent assay（ELISA）. Results After 30-minute co-culture of LAD2 cells and SEB, MCs showed larger size, obscure boundaries, increased number of protuberances on the cell surface and decreased refractivity, with a radial burr fin-like appearance. After 30-minute co-culture of LAD2 cells and SEB at different concentrations of 0.01, 0.1, 1, 10 and 100 μg/ml, the concentrations of tryptase in the culture supernatants were 4.116 ± 0.651, 5.344 ± 0.874, 3.806 ± 0.459, 1.309 ± 0.247, 0.310 ± 0.199 ng/ml respectively. Additionally, the tryptase levels were significantly higher in the 0.01-, 0.1-, 1-μg/ml SEB groups than in the negative control group（1.538 ± 0.490, all P 〈 0.05）, and gradually decreased along with the increase of SEB concentrations. The histamine levels in the 0.01-, 0.1-, 1-, 10- and 100-μg/ml SEB groups were 242.409 ± 63.915, 522.491 ± 73.466, 550.926 ± 84.466, 334.397 ± 33.640, 226.527 ± 5.678 ng/ml respectively. In the 0.01-, 0.1-, 1-μg/ml SEB groups, the levels of histamine released from MCs were gradually increased along with the increase of SEB concentrations, and were significantly higher than those in the negative control group （146.436 ± 3.100, all P 〈 0.05）. However, with the continued increase of SEB concentrations, the histamine levels gradually decreased. Conclusion SEB can directly activate MCs by a non-IgE mediated mechanism, followed by morphologic changes of MCs and release of tryptase and histamine.