烟曲霉菌提取物对人支气管上皮细胞Muc5ac表达的影响

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

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、20mg/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Muc5acmRNA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。

AIM： To investigate the effects of Aspergillus fumigatus extract （AFE） on the expressions of MucSac 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 MucSac 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 MucSac 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 MucSac production by 16HBE- 14o. Heat- treated AFE, which lost protease activities, exerted no effect on MucSac 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.