鞭毛蛋白致肺微血管内皮细胞炎症诱发共培养肺泡上皮细胞炎症的级联放大效应与信号传导通路

Inflammation cascade effect and signaling transduction pathway of alveolar epithelial cells co-cultured with pul-monary microvascular endothelial cells treated with flagellin

目的探讨鞭毛蛋白感染后炎症级联放大效应与信号传导通路。方法建立Transwell共培养体系，将人肺微血管内皮细胞株（ human pulmonary microvascular endothelial cells, HPMECs ）接种于Transwell小室的下层，培养2h后，将人Ⅱ型肺泡上皮细胞A549细胞株接种于小室的上层与HPMECs共培养15d。实验分为3组：HPMECs与A549细胞共培养空白对照组、鞭毛蛋白（2μg／mL）感染HPMECs再与A549细胞共培养组（实验组I）、HPMECs加DAPT（Notch信号阻断剂，终浓度10μmol／L）预处理再行鞭毛蛋白感染与A549细胞共培养组（实验组Ⅱ）。用ELISA法检测各组HPMECs和A549细胞培养上清液TNF-α蛋白表达，检测HPMECs上清液Notchl蛋白表达水平。结果与共培养空白对照组比较，鞭毛蛋白感染HPMECs后，共培养HPMECs和A549细胞上清液TNF-α蛋白[（11．45±1．59）pg／mLvs（6．13土0．86）pg／mL，（P〈0．01）、（9．93±1．46）pg／mLvs（5．895：0．83）pg／mL，（P〈0．01）]和HPMECs上清液中Notchl蛋白[（7．03±1．06）pg／mL坩（5．39±0．76）pg／mL，（P〈0．05）]表达水平皆明显升高，提示鞭毛蛋白引起HPMECs炎症，且向A549细胞传导级联放大，而HPMECs使用Notch信号阻断剂处理后，HPMECs上清液Notchl蛋白表达水平明显降低[（3．78±0．53）pg／mLvs（7．03±1．06）pg／mL，（P〈0．01）]，共培养A549细胞上清液TNF-α蛋白表达水平也显著下降[（7．47±1．05）pg／mL坩（9．93±1．46）pg／mL，（P〈0．05）]，表明HPMECs炎症反应经过Notch信号通路传导向A549细胞级联放大。结论鞭毛蛋白感染肺微血管内皮细胞能引起炎症反应，并可能经过Notch信号通路传导向肺泡上皮细胞级联放大。

Objective To explore inflammation cascade effects of flagellin infection and signaling transduction pathway. Methods A Transwell coculture system of human pulmonary microvascular endothelial cell （HPMECs） and A549 cells was constructed. HPMECs, which was put into the lower chamber and cultured for 2 hours, were co-cultured with A549 cells put in the upper chamber for 15 days. 3 groups were set as below： HPMECs were co-cultured with A549 cells （control group）, HPMECs infected with flagellin （ a final concentration of 2μg/mL ） were co-cultured with A549 cells （experimental group I ） and flagellin-infected HPMECs treated with DAPT （inhibitor of notch signaling, a final concentration of 10 μmol/L） were co-cultured with A549 cells （experimental group Ⅱ）. The proteins of TNF-α and Notchl in supernatant of HPMECs and A549 cells in all cultured groups were evaluated by ELISA assay, respectively. Results Compared with the control group, the levels of proteins of TNF-α and Notch1 in supernatant of HPMECs [（11.45±1.59） pg/mL vs （6.135=0.86） pg/mL, （P〈0.01）, （7.03±1.06） pg/mL vs （5.39±0.76） pg/mL, （P〈0.05）] and supernatant levels of TNF-a in A549 cells [（ 9.93 ± 1.46 ） pg/mL vs （ 5.89± 0.83 ） pg/mL, （P 〈 0.01 ）] in all cultured groups were significantly increased in the experimental group I. These results indicated that flagellin caused HPMECs inflammation, which was enlarged and transducted to the co-cultured A549 cells, whereas this phenomenon was inhibited by using DAPT, indicating that A549 cells inflammation induced by flagellin-infected HPMECs was transducted through Notch signaling pathway. Con- chsion Flagellin infection can cause inflammation of pulmonary microvascular endothelial cells, which may be enlargedand transducted to alveolar epithelial cells through the Notch signaling pathway.