烟曲霉菌提取物对人支气管上皮细胞Muc5ac表达的影响

目的： 探讨烟曲霉菌对呼吸道上皮细胞的黏液蛋白Muc5ac表达的影响及其可能机制。方法: 分别应用不同浓度(0、8、16、20 mg/L) 的烟曲霉菌提取物（AFE）作用体外培养人支气管上皮细胞16HBE-14o不同的时间。实验还应用热处理的AFE（HT-AFE）、丝氨酸蛋白酶抑制剂（抑肽酶）和蛋白激酶激活受体-2（PAR-2）阻断剂（FSLLRY-NH2）。应用细胞免疫组织化学和酶联免疫法(ELISA)检测细胞Muc5ac蛋白量，同时应用RT-PCR检测Muc5ac mRNA 的表达。结果: 正常未给予AFE刺激培养条件下的对照组细胞仅表达少量Muc5ac，而给予不同浓度的AFE作用后，细胞合成和分泌Muc5ac的量明显高于对照组（P＜0.01），并呈明显的时间和浓度依赖性。丝氨酸蛋白酶抑制剂和PAR-2特异性阻断剂可以明显抑制AFE的上述作用。另外，热处理的AFE蛋白酶活性完全灭活，不能促进细胞Muc5ac表达增加。结论: AFE依赖其蛋白酶活性激活受体PAR-2，促进呼吸道上皮细胞Muc5ac表达增加，使黏液分泌增加。

Effects of Aspergillus fumigatus extract on expression of Muc5ac in human bronchial epithelial cells

AIM: To investigate the effects of Aspergillus fumigatus extract (AFE) on the expressions of Muc5ac in human bronchial epithelial cells and its possible mechanism. METHODS: Human bronchial epithelial cells (16HBE-14o) were cultured in vitro, which were exposed to different concentrations of AFE (0, 8, 16, 20 mg/L) for different times. In order to explore the mechanisms, heat-treated AFE, serine protease inhibitors (aprotinin) and protease-activated receptor-2 (PAR-2) antagonist (FSLLRY-NH2) were used. The production and release of Muc5ac in different intervals were tested by immunohistochemistry and ELISA. The expression of Muc5ac mRNA was measured by RT-PCR. RESULTS: In normal control group, only a few Muc5ac was detected. In the experimental groups with AFE exposure, cells produced more Muc5ac compared to normal control group (P＜0.01), which were positively related to the exposure time or the concentration of AFE. Aprotinin and PAR-2 antagonist (FSLLRY-NH2) inhibited the effect of AFE on Muc5ac production by 16HBE-14o. Heat-treated AFE, which lost protease activities, exerted no effect on Muc5ac production and mRNA expression. CONCLUSION: AFE, depending on its protease activity, activates PAR-2 and causes airway epithelial cells to produce and release more Muc5ac, which may contribute to deterioration of asthma.