【作者】 张桂敏；

【导师】 杨洁；

【作者基本信息】 天津医科大学 ， 免疫学， 2011， 博士

【摘要】 目的：鼻息肉是鼻腔外侧壁粘膜突入鼻腔形成的新生物,发病机制不明,其本质是粘膜的慢性持续性炎症。白细胞介素18(IL-18)是近年来发现的前炎性细胞因子,在感染、炎症、自身免疫性疾病、组织的炎性损伤等过程中发挥非常重要的调节作用。p100蛋白是一种多功能蛋白,它可与多种蛋白转录因子结合,广泛的存在于各种组织细胞。然而,查阅文献,未见IL-18和p100蛋白在鼻息肉中的表达情况的报道。由此,我们提出疑问：IL-18和p100在鼻息肉中的表达情况如何呢?二者与鼻息肉的发病是否有关系呢?二者在鼻息肉的发病机制中到底扮演什么样的角色呢?其深入的分子机制又是如何?此课题我们检测IL-18和p100在鼻息肉中的表达情况,并进一步探讨二者在鼻息肉形成过程中可能的作用机制。方法：第一部分：IL-18等细胞因子及p100蛋白在鼻息肉中的表达与定位。收集鼻息肉和正常鼻粘膜临床标本,将其分为两部分,一部分制作石蜡病理切片,进行HE及免疫组化染色,观察鼻息肉炎细胞构成及浸润情况,观察IL-18、p100蛋白在鼻息肉中的表达情况。另一部分液氮储存,匀浆并变性蛋白后进行Western Blotting检测,在蛋白水平进一步的验证IL-18、p100蛋白在鼻息肉中的表达情况。第二部分：IL-18在鼻息肉形成过程中可能作用机制的研究。这部分实验中,我们以A549细胞系作为呼吸道上皮细胞的代表,以炎症因子LPS,细胞因子IL-4刺激A549细胞,观察IL-18的表达情况以明确上皮细胞在炎症状态下是否会分泌IL-18等细胞因子以及上皮细胞分泌的细胞因子之间是否会相互调控,以进一步的证明IL-18参与了鼻息肉形成的病理机制,且上皮细胞可能是鼻息肉病理机制发生的"initiator"。第三部分：p100在鼻息肉形成过程中可能作用机制的。在这部分实验中,我们将实验分成两个部分,(1)p100是否参与IL-4/STAT6和LPS/TLR4信号通路的研究；(2)p100是否参与增殖和细胞周期的研究。在第一部分实验中我们的技术路线是：在细胞水平,以LPS, IL-4刺激A549细胞,观察IL-8, IL-6等细胞因子,STAT6, p100的表达变化情况。在动物水平,我们以OVA诱导小鼠哮喘模型以模拟IL-4/STAT6信号通路,以LPS诱导急性肺炎模型模拟LPS/TLR4信号通路,取小鼠肺组织,观察支气管上皮细胞p100表达情况。在第二部分实验中我们又分为两个小的实验①p100与增殖状态关系的初步研究和②p100与细胞周期相关转录因子c-Myc的关系的研究,实验①的技术路线是取小鼠不同增殖状态的组织和不同增殖状态的外周血细胞检测p100蛋白的表达水平。实验②的技术路线是第一,我们培养裂解hela细胞,分别以p100抗体钓取c-Myc蛋白,以c-Myc抗体钓取p100蛋白,WB检验钓取结果,以明确p100蛋白是否与c-Myc蛋白相结合。第二,以不同的质粒转染hela细胞,建造p100过表达,和p100抑制的细胞模型,检测c-Myc的表达情况,以视p100表达是否对c-Myc蛋白表达产生影响。结果：第一部分：我们发现IL-18、 IL-4、 IFN-y在正常鼻粘膜及鼻息肉的上皮层、腺体及间质的炎性细胞中均有表达,且在鼻息肉中表达增加,在嗜酸粒鼻息肉中表达增加更明显,成熟型IL-18仅在鼻息肉中表达,而正常鼻粘膜中缺如。我们还发现p100蛋白在鼻息肉上皮层和腺体中也大量表达,但在80%的嗜酸粒细胞中表达缺失。第二部分：我们证实IL-18在上皮细胞系A549中表达,且在LPS刺激下表达增加,成熟型IL-18仅在LPS刺激的A549细胞中表达,而正常A549中缺如。我们还发现IL-4能减弱IL-18在上皮细胞系A549中表达。细胞因子在上皮细胞的表达也可相互调控。第三部分：p100蛋白参与炎症的机制可能是参与了IL-4/STAT6信号转导通路,而没有参与LPS/TLR4信号通路诱导的细胞因子生成过程。另外,p100蛋白可能与细胞周期密切相关,其参与细胞周期可能不是通过c-Myc途径,而是通过其它的信号通路。结论：IL-18、p100正常鼻粘膜及鼻息肉中均大量表达。IL-18在鼻息肉中表达增加,成熟型IL-18仅在鼻息肉中表达,而正常鼻粘膜中缺如。IL-18在鼻息肉的形成过程中可能发挥重要的作用。鼻粘膜上皮细胞可在致炎因素的刺激下分泌大量的细胞因子如IL-18、 IL-4、IFN-r等,鼻上皮细胞是接触抗原的第一道屏障,可能是鼻息肉等疾病发生发展的‘’’initiator"。 p100蛋白通过IL-4/STAT6信号转导通路参与了鼻息肉的炎症机制,p100蛋白可能与细胞周期密切相关,参与了鼻息肉的增生,在鼻息肉的形成过程中起非常重要作用。更多还原

【Abstract】 Objectives:Nasal polyposis is a chronic inflammatory disease of the nasal mucosa. The etiology and mechanisms of the formation of nasal polyps are still not clear. Interleukin (IL)-18is a novel proinflammatory cytokine that plays important roles in regulating immune inflammatory responses。 p100protein on the other hand can combine with many kinds of transcription factors. It is a ubiquitously and highly conserved protein expressed in a wide range of eukaryotes from yeasts to human. However, the presence of IL-18and p100protein in human nasal mucosa and their roles in the inflammatory process of nasal polyps has not been studied yet. In the present study, we investigate the expression of IL-18and p100protein in human nasal mucosa and nasal polyps, and their potential function in the formation of nasal polyps.Methods:Part I:The expression of IL-18and p100in nasal polyps. Nasal mucosa of control samples and nasal polyp samples were obtained. Each sample was divided into two parts. One part was homogenized immediately and quick-frozen for Western blot analysis. The other was fixed in a10%neutral formaldehyde solution and embedded in paraffin for hematoxylin-eosin staining and immunohistochemistry.Part II:The potential role of IL-18in the formation of nasal polyps. A549cells, which is often used as representative of respiratory epithelial cells, were treated with LPS (1μg/ml) or IL-4(50ng/ml), to some extent, mimic infection and inflammation. Then detect the expression of IL-18in order to verify that epithelial cells can express more IL-18and IL-18can be regulated by other cytokines such as IL-4. Thus, evaluation of their expression will provide new insights into the pathophysiological roles of IL-18in the pathogenesis of nasal polyp formation, and in the potential role of epithelial cells in this process.Part III:The potential role of p1OO in the formation of nasal polyps. In this part, we divide the study into two parts. In the first part, we treat A549cells with LPS and IL-4to mimic LPS/TLR4and IL-4/STAT6signal pathways respectively. Then, we detect the expression of IL-8, IL-6, STAT6and p1OO. We also detect the expression of p100protein in the bronchial epithelial cell of mouse lung which were treated with LPS and OVA also to mimic LPS/TLR4and IL-4/STAT6signal pathway. In the second part, we want to study the relationship between pi00protein and proliferation. We also divide this study into two parts:①we detect the expression of p100protein in mouse tissues with different proliferation status and the expression of p100protein in different peripheral blood cells.②we detect if p100protein can combine with c-Myc protein by COIP and detect if the expression of p100protein can influence the expression of c-Myc protein.Results:Part I:We observed that in both the normal sinus mucous and nasal polyps, IL-18, IFN-y and IL-4immunoreactivity was observed in the epithelium, submucosal glandular cells and the inflammatory cells. Moreover, the intensity of the IL-18, IFN-y and IL-4bands in the eosinopbilic nasal polyps were higher than that of non-eosinophilic nasal polyps. We also observed p100protein is expressed in the epithelium, submucosal glandular cells in nasal polyps, but absent in80%of eosinophils.Part II:We found that IL-18is expressed in A549cells and the expression of IL-18was increased after LPS treatment. The18-kDa mature form of IL-18could only be detected after LPS treatment, but was absent in the control groups. We also found that IL-18expression was lower after IL-4treatment in A549cells. Cytokines from airway epithelial cells can regulate each other.Part III:pi00protein take part in IL-4/STAT6signal pathway but not in LPS/TLR4signal pathway in the process of inflammation. Further more, p100protein maybe related with cell cycle and tissues proliferation. And our result showed that p100protein take part in the cell cycle not in c-Myc pathway but may be in other signal pathway.Conclusions:IL-18and p100protein expressed in normal nasal mucous and in nasal polyps. Since mIL-18only can be detected in nasal polyps, IL-18play an important role in the formation of nasal polyps. Nasal epithelial cells maybe the "initiator" in the formation of nasal polyps since they are the first physical barriers against airborne particles from the environment and can release lots of cytokines such as IL-18、 IL-4、 IFN-yand so on. p100protein takes part in IL-4/STAT6signal pathway but not in LPS/TLR4signal pathway in the process of inflammation, p100protein is related with the cell cycle and tissues proliferation and plays an important role in the formation of nasal polyps.更多还原