Toll样受体4在人肺泡上皮细胞株中的表达及其在细胞炎症反应中的作用

Expression of Toll-Uke receptor 4 in human alveolar epithelial cells and its role in cellular inflammation

目的明确人Ⅱ型肺泡上皮细胞是否表达Toll样受体4（TLR4），探讨TLR4在慢性阻塞性肺疾病（COPD）气道炎症中的作用。方法将人Ⅱ型肺泡上皮细胞系A549细胞分为正常对照组、转染腺病毒E1A质粒（E1A^＋组）和转染不含有腺病毒E1A的空白质粒组（E1A^-组），分别以不同浓度的脂多糖（LPS，0、0．1、1、10μg／ml）、白细胞介素1β（IL-1β，0、0．1ng／ml）、香烟提取物（CSE，0.10％、20％、40％）刺激。刺激后12、24h，用反转录聚合酶链反应技术检测各组细胞IL-8 mRNA和TLR4 mRNA的表达，用蛋白质印迹技术测定核因子κB（NF—κB）亚单位P65蛋白的表达。结果10μg／ml LPS、0．1ng／ml IL-1β或20％CSE刺激24h后，E1A^＋组IL-8 mRNA表达量（2．82、1．87、4．70）明显高于正常对照组（0．95、0．78、1，02，均P〈0．05）和E1A^-组（0．97、0．81、1．12，均P〈0．05）。10μg／ml的LPS刺激后12、24h，E1A^＋组细胞TLR4 mRNA表达水平分别为4．52、7．99，明显高于正常对照组（1．91、3．81，均P〈0．05）和E1A^-组（2．00、3．88，均P〈0．05）；IL-1β也可增加TLR4 mRNA表达。CSE刺激对各组细胞TLR4 mRNA表达水平均无明显影响。3种刺激因子均可引起各组细胞NF-κB亚单位P65蛋白表达水平增高。结论人Ⅱ型肺泡上皮细胞表达TLR4，LPS、IL-1β引起IL-8释放可能与TLR4介导的NF-κB激活有关。

Objective To investigate if type Ⅱ alveolar epithelial cells express Toll-like receptor 4 （TLR4） and to investigate the role of TLR4 in airway inflammation of chronic obstructive pulmonary diseases （COPD）. Methods A549, the line of human type Ⅱ alveolar epithelial cells were cultured and divided into 3 groups ： normal control group, E1A ^＋ group transfected with adenovirus E1A plasmid, E1A - group transferred with blank plasmid without adenovirus E1A. Lipopolysaccharide （LPS） of the concentrations of 0, 0.1, 1, and 10 μg/ml, IL-1 β of the concentrations of 0, and 0. 1 ng / ml, and cigarette smoking extract （CSE） of the concentrations of 0, 10%, 20 %, and 40% were used to stimulated the 3_549 cells for 12 and 24 h. Reverse transcription polymerase chain reaction was used to detect the mRNA expression of IL-8 and TLR4. Western blotting was used to detect the protein expression of nuclear factor KB （NF-κB） subunit 1065. Results Twenty-four hours after the stimulation of 10 μg/ml LPS, 0. 1 ng/ml IL-1β, and 20% CSE, the IL-8 mRNA expression of the E1A^＋ group was 2.82, 1.87, and 4.70 respectively, all significantly higher than those of the normal control group （0. 95, 0.78, and 1.02 respectively, all P 〈 0.05 ） and those of the E IA - group （0.97, 0.81, and 1.12 respectively, all P 〈 0.05 ）. Twelve and twenty-four hours after the stimulation of 10 μg/ml of LPS, the TLR4 mRNA expression of the E1A ^＋ group were 4.52 and 7.99, beth significantly higher than those of the normal control group （1.91 and 3.81 respectively, both P 〈 0.05 ） and those of the E1A - group （2.00 and 3.88 respectively, both P 〈 0. 05 ）. IL-1 β increased the expression of TLR4 mRNA too, but CSE did not change the expression of TLR4 mRNA in all these groups. LPS, IL-1 β, and CSE all increased the expression levels of NF-KB subunit P65 protein. Conclusions Pulmonary type Ⅱ epithelial cells express TLR4. LPS and IL-1 β up-regulate the release of IL-8 which may be mediated via the activation of NF-κB induced by TLR4.