组胺对肺上皮细胞Toll样受体表达上调作用.doc

组胺对肺上皮细胞Toll样受体表达上调作用 　 作者： 侯一峰，周艳春，郑晓璇，王海燕，傅意玲，方泽曼，何韶衡 【关键词】 组胺；肺/细胞学；上皮细胞；Toll样受体 【Abstract】 AIM: To investigate the upregulatory effect of histamine on Tolllike receptor (TLR) expression in pulmonary epithelia cells. METHODS: TLR1TLR10 mRNA expressions were determined by RTPCR in resting A549 cells, and by realtime quantitive PCR in the cells following stimulation with histamine at various concentrations and at various periods of time. Furthermore, the upregulatory effect of histamine on TLR3 was examined by flow cytometry. RESULTS: TLR1TLR10 mRNA were constitutively expressed in A549 cells. Histamine significantly enhanced TLR3 expression at both mRNA and protein levels in a concentration and timedependent manner. This effect of histamine was blocked by the H1 receptor antagonist. CONCLUSION: TLR3 expression can be upregulated by histamine in A549 cells, which suggests that excessive histamine can enhance the sensitivity of pulmonary epithelium to virus infection when the allergic diseases exist in the respiratory tract. 【Keywords】 histamine, lung/cytology; epithelial cells, Tolllike receptor 【摘要】 目的： 观察组胺对肺上皮细胞中Toll样受体（TLR）表达的影响. 方法： 体外培养人肺上皮细胞株A549，用RTPCR和实时定量PCR检测静息状态或组胺作用下的A549细胞中TLR1～TLR10 mRNA的变化，并用流式细胞术进一步确定组胺对TLR3表达的调节作用. 结果： A549细胞有TLR1～TLR10 mRNA的组成型表达，组胺对TLR3 mRNA和蛋白的上调作用具有时间和剂量依赖性，并且H1受体阻断剂可抑制组胺对TLR3的上调作用. 结论： 组胺可上调肺上皮细胞中TLR3的表达，提示当呼吸道存在变态反应性病变时，增多的组胺可能会增加肺上皮细胞对病毒感染的敏感性. 【关键词】 组胺；肺/细胞学；上皮细胞；Toll样受体 呼吸道病毒感染是支气管哮喘急性发作的一个重要诱因［1］，而有关哮喘急性发作时呼吸道对病原菌感染的敏感性及其机制目前尚不清楚. Toll样受体（Toll likereceptor, TLR）是一类在天然免疫识别中具有重要作用的模式识别受体，目前在人体内至少已发现TLR家族中的10个成员(TLR1～TLR10). TLR广泛表达于各类组织细胞中，且许多炎性介质可影响TLR的表达［2］. 组胺是一种在支气管哮喘中发挥重要作用的介质，有研究显示：组胺可上调血管内皮细胞中TLR2和TLR4的表达［3］. 本研究旨在通过观察组胺对肺上皮细胞中TLR表达的影响，探讨哮喘急性发作与呼吸道感染之间的关系. 1材料和方法 1.1材料人肺上皮细胞株A549购自美国ATCC(American Type Culture Collection)，胎牛血清、DMEM培养基购自Hyclone公司. TRIzol RNA提取试剂、ProofStart DNA聚合酶和Quanti Tet TM SYBR Green PCR Kit购自Invitrogen公司,RNA酶抑制剂和MMLV逆转录酶均购自Promega公司. 人TLR1～TLR10及βactin的PCR引物由上海生工生物工程公司合成. PCR产物回收试剂盒购自上海生工生