【作者】 周建娅；

【导师】 唐法娣；

【作者基本信息】 浙江大学 ， 药理学， 2003， 硕士

【摘要】 前言：呼吸系统疾病大部分与免疫、炎症反应密切相关，如急慢性肺损伤，哮喘、慢性阻塞性肺病(COPD)，其发病率和死亡率日趋升高。核因子-κB(NF-κB)作为一种普遍存在的多功能的转录因子，是细胞内多种信号转导途径的汇聚点，不仅参与介导了免疫应答、细胞应激、凋亡和增殖等多种相关基因的表达调控，而且在调节哮喘、肺损伤、COPD等肺部疾病的有关炎症反应基因中起关键作用。参与肺部炎症性疾病的细胞因子种类极多，信号网络极为复杂，如果我们能在一个较高的水平对产生炎性因子的总体环节进行调控，而不是对上游信号通路孤立地分而治之，将成为寻找新型抗炎药物的新方向。故我们把NF-κB作为抗炎靶点，以抑制NF-κB的病理性过度激活为目标，寻找较特异的NF-κB活性抑制剂，对肺部炎症性疾病中调控炎症反应的关键环节进行干预，将有广阔深远的应用前景。 从蓝桉叶和樟树叶中提取的挥发油总称桉叶油，其中从蓝桉叶中提取的又称蓝桉油，桉叶油和蓝桉油主要含有1.8-桉油素、α-蒎烯等，目前研究发现桉叶油具有抗炎症的作用，但其抗炎分子机制仍不明确，鉴于NF-κB在肺部免疫，炎症中的重要地浙江大学硕士学位论文 肺部炎症模型中校因子一d的改变及蓝按油和a＆烯对其核转位活化的影响位，故我们以NF－h作为抗炎症效应的靶标来研究蓝按油的抗炎机制。目的：建立脂多糖（LPS）THP－l细胞致炎模型、小剂量LPS气管滴入及长期二氧化硫（SO。）吸入诱发的大鼠慢性肺部炎症模型，观察NF－h的活化状态，探讨蓝按油及其主要成分a．菠烯对以上三模型中NF．h核转位活化的影响。方法：采用人血单核细胞株THP．l、SD大鼠为实验对象，建立脂多糖（LPS）THP．l细胞致炎模型、小剂量LPSu00 pg）气管滴入及长期SO。吸入诱发的大鼠慢性肺部炎症模型，并各自以蓝按油及a．获烯为给药组。运用间接免疫荧光细胞化学法结合激光扫描共聚焦显微镜（LSCM）测定THP－l细胞、大鼠支气管肺泡灌洗液中肺泡巨噬细胞（Pulmonary alveolar macrophages，PAMs）以异硫氰酸荧光素（FITC）绿色荧光标记的NF－h p65亚单位（NFth／p65）定位；运用低温和抑蛋白酶条件下，低渗裂解细胞膜提取胞浆蛋白，继而高渗裂解核膜提取核蛋白，Folin酚法定量蛋白，再以免疫印迹电泳法（Western．blot）、双抗体夹心酶联免疫吸附实验法（SandwichELISA）分析细胞核内NF－h／p65水平、胞浆内I。Ba水平变化，并进行半定量分析。统计学处理分析采用SPSS 11刀1 统计软件包，统计数据以二d表示，显著性检验用单因素方差分析。结果：1．免疫荧光LSCM分析显示：＠正常状态的THP1细胞，大鼠PAMS中NF七B／p65荧 光标记主要分布于胞浆区，核区很少，经LPS 30 min刺激的THP－l细胞、经小剂 量LPS气管滴入3 W后的大鼠PAMS中NF－h／p65荧光标记浓集于核区，胞浆区少 见。②经蓝按油或a一菠烯预处理的Tm－l细胞，再以LPS 30 min刺激，NF－h／p65 荧光标记则仍主要位于胞浆区；经小剂量LPS气管滴入，3 W内蓝按油给药的大 鼠PAMS中NF－h／p65荧光标记也仍主要位于胞浆区。2．Western七lot分析显示：＠正常THPl细胞核蛋白中有低水平的NF－loB／p65表达， 2浙江大学硕士学位论文 肺部炎症模型中校因于一d的改变及蓝按油和a菠烯对其核转位活化的影响 经LPS 30 min刺激的THP＊细胞核内NF－h／p65表达明显增高（P＜0刀5人 正常大 鼠肺组织细胞核蛋白中有低水平的NF－oh／p65表达，经小剂量LPS气管滴入及长 期SO。吸入的大鼠肺组织细胞核内NF－h／p65表达明显增高。＠蓝按油或a一菠烯 门、10、100 pg·L’，30min）预处理可以抑制LPS 30min刺激诱导的THP上细胞核 内NF啦／p65的高水平表达，且随着蓝按油或a一菠烯剂量的增加，NF－h／p657k平 相对降低，经统计分析，呈良好的剂量依赖关系，其中蓝按油 10 pg·L”’和 100 叩·L’组、队获烯100 Hg·L’组，有统计学差异（p＜0刀5）；经小剂量Lps气管滴入， 3w内持续蓝按油Q0、100、300mg，kg‘）给药的大鼠肺组织细胞核内NF俯／p65 表达比模型组降低，且呈一定的剂量依赖效应。长期SOZ吸入，SW内持续蓝按油 （10 mg·kg－’）给药的大鼠肺组织细胞核内NF四／p65表达也比模型组明显降低。 ③o一派烯门、10、100 pg·L－’，30mifl）对正常培养状态THpl细胞胞核中的 NF一h／p65未见明显影响（HO刀5）。＠a一派烯（、10、100 pg·L”l，30 min）预 处理，可抑制LPS诱导胞浆内Itha降解，且经统计分析，呈剂量依赖关系，其中 a一菠烯 100 卜g·L－’组有统计学差异（P＜0．05）。3．Sandwich ELISA分析显示：SO。吸入模型NF－h的A。。。nm值与正常大鼠比较明显升 高（P＜0．05X而蓝按油100 mg·kg”’组NFth／p65的A巧＊m值与模型组比较明显下 降（P＜0刀5）。结论：1．LPS体外致炎模型中LPS刺激能引起THP－1细胞NF－IcB核转位活化?更多还原

【Abstract】 Introduction: A majority of pulmonary diseases, such as lung hurt, asthma, chronic obstructive pulmonary disease （COPD）, have strong affinity with immune and inflammatory responses. Nuclear factor-KB （NF-?B） is an important pluripotent transcription factor. It’s gene products include cytokine, chemokines, cell adhesion molecules, and immunoreceptors. NF-?B plays a key role in immune and inflammatory responses. Therefore, modulation of NF-KB activation may provide a direct way of inhibiting inflammatory mediators. Directing drug discovery efforts towards NF-KB activation rather than towards any one of its many target genes could produce a much greater therapeutic benefit by inhibiting expression of the constellation of NF-?B-induced pro-inflammatory genes.Eucalyptus globulus oil shows particular anti-inflammation properties, which include two main components: 1.8-cineole and a-pinene. However, the molecular of the anti-inflammation activity of eucalyptus globulus oil is still not well understood. Since NF-KB is a central mediator of inflammatory responses, we hypothesize that NF-KB is a target for eucalyptus globulus oil-mediated anti-inflammation properties.Objective: Set up Lipopolysaccharide （LPS）-induced THP-1 cells inflammation model and rats’ chronic lung inflammation models induced respectively by LPS-airway-injection and sulfur dioxide （S02）-airway-inhalation. Detect the activation of nuclear factor-?B. Discuss the effects of Eucalyptus globulus oil and its main component, a-pinene on the activation of nuclear factor-KB of the three above models.Methods: THP-1 cells were incubated with a series of doses of drugs before being stimulated with LPS （1 mg.L-1, 30 min）. SD rats were injected with LPS or inhaled with SO₂ from airway and took a series of doses of drugs orally. The location of NF-?B p65 subunit （NF-?B/p65） in THP-1 cells and pulmonary alveolar macrophages （PAMs） was detected by indirect immunofluorescence and laser scanning confocal microscope（LSCM）. The expression of NF-?B/p65 in nuclei and I?Ba in cytoplasm were measured by Western -blot analysis or Sandwich ELISA analysis. Semi-quantitative analysis of Western -blot was measured. The data are presented as mean ?SD and compared with one-way ANOVA analysis using SPSS statistical program.Rusluts:1. Indirect immunofluorescence under LSCM showed: ?The majority of FITC-label NF-?B/p65 located in the cytoplasm of control THP-1 cells or PAMs, whereas, being stimulated with LPS the majority of NF-?B/p65 located in nuclei. ?No such fluorescence were seen in the nuclei of the cells pretreated with eucalyptus globulus oil or a-pinene.2. Western-blot analysis showed: ?A low level expression of NF-?B/p65 in nuclei was measured by in control THP-1 cells or rat lung tissue, but a obvious induction of NF-?B/p65 nuclear translocation was observed in THP-1 cells being stimulated with LPS （P<0..05） or in rat lung tissue being stimulated with LPS, SO₂ from airway. （2） Eucalyptus globulus oil and a-pinene （1?10?100 礸.L-1, 30 min） pretreatment decreased the NF-?B/p65 nuclear translocation in LPS-stimulated THP-1 cells, and these effects were dose-dependent （P<0.05）. Eucalyptus globulus oil decreased the NF-?B/p65 nuclear translocation in the rat lung tissue being .stimulated with LPS, SOifrom airway. (? Only treatment with a-pinene （1?10?100 ng.L-1, 30 min） did not significantly affect on the NF-?B/p65 nuclear translocation in normal THP-1 cells （P>0.05）. ?The I?Ba protein level in LPS-stimulated THP-1 cells markedly decreased at 30 min. The LPS-induced degradation of I?Ba was blocked by a-pinene （P<0.05）.3. Sandwich ELISA analysis showed: A45onm of NF-?B/p65 in the rat lung tissue being inhaleded with SO2 from airway was markedly increased compared with control, but this effect was decreased by treated with eucalyptus globulus oil （P<0.05）.Conclusions:1. In LPS-induced THP-1 cells inflammation model, LPS could stimulate the NF-icB activation. In the rats’ chronic lung inflammation models induced respe更多还原