IL-18和p100蛋白在鼻息肉形成过程中潜在作用机制的研究

发布时间：2018-07-12 11:28

本文选题：Il-18 + p100　；参考：《天津医科大学》2011年博士论文

【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。
[Abstract]:Objective: nasal polyps are new organisms that penetrate the nasal cavity of the lateral wall of the nasal cavity. The pathogenesis is unknown and its essence is chronic persistent inflammation of the mucous membrane. Interleukin 18 (IL-18) is a proinflammatory cytokine discovered in recent years. It plays a very important role in infection, inflammation, autoimmune diseases and inflammatory injury of tissues. .p100 protein is a multifunctional protein, which combines with a variety of protein transcription factors and exists widely in various tissue cells. However, there is no report on the expression of IL-18 and P100 proteins in nasal polyps. Therefore, we ask questions: how is the expression of IL-18 and P100 in nasal polyps? The two and the nasal polyps What is the relationship between the pathogenesis of polyps? What is the role of the two in the pathogenesis of nasal polyps? How is its deep molecular mechanism? We examine the expression of IL-18 and P100 in nasal polyps, and further explore the possible mechanisms of the action of the two in the formation of nasal polyps.
Method:
The first part: the expression and localization of IL-18 and P100 protein in nasal polyps. The nasal polyps and normal nasal mucosa were collected and divided into two parts. Part of the paraffin pathological sections were made, HE and immunohistochemical staining were used to observe the composition and infiltration of nasal polyps, and the IL-18 and P100 protein were observed in the nasal polyps. Another part of liquid nitrogen storage, homogenate and denatured protein were detected by Western Blotting, and the expression of IL-18 and P100 protein in nasal polyps was further verified at the protein level.
The second part: the possible mechanism of IL-18 in the formation of nasal polyps. In this part, we use the A549 cell line as the representative of the respiratory epithelial cells. We use the inflammatory factor LPS and the cytokine IL-4 to stimulate the A549 cells, and observe the expression of IL-18 to determine whether the epithelial cells secrete IL-18 and other cells in the inflammatory state. Whether the factors and the cytokines secreted by the epithelial cells are regulated by each other, in order to further demonstrate that IL-18 is involved in the pathogenesis of nasal polyps, and that epithelial cells may be "initiator" in the pathogenesis of nasal polyps.
The third part: the possible mechanism of P100 in the formation of nasal polyps. In this part, we divide the experiment into two parts, (1) whether P100 participates in the study of IL-4/STAT6 and LPS/TLR4 signaling pathways; (2) whether P100 participates in the study of proliferation and cell cycle. In the first part of the experiment, our technical route is in cells Level, using LPS, IL-4 to stimulate A549 cells, observe the changes in the expression of IL-8, IL-6 and other cytokines, STAT6, P100. At animal level, we induced the mice asthma model by OVA to simulate the IL-4/STAT6 signaling pathway, and the acute pneumonia model was induced by LPS to simulate the LPS/TLR4 signaling pathway, the lung tissue of mice was taken and the expression of the bronchial epithelial cell P100 expression was observed. In the second experiment, we divide into two small experiments: preliminary study on the relationship between P100 and proliferation state and the relationship between P100 and cell cycle related transcription factor c-Myc. The technical route of the experiment is to take the form of mice with different proliferating States and peripheral blood cells with different proliferation states to detect the P100 protein The technical route of Experiment 2 is the first. We cultivate lysis HeLa cells, catch c-Myc protein with P100 antibody, catch P100 protein with c-Myc antibody, and catch the result by WB test to determine whether P100 protein is combined with c-Myc protein. Second, transfect HeLa cells with different plasmids, construct P100 overexpression, and P100 inhibition cell model The expression of c-Myc was detected to determine whether P100 expression has an effect on the expression of c-Myc.
Result:
The first part: we found that IL-18, IL-4, IFN-y were expressed in the epithelial layer of normal nasal mucosa and nasal polyps, in both glands and interstitial inflammatory cells, increased in nasal polyps and increased in eosinophilic nasal polyps. Mature IL-18 was only expressed in nasal polyps, while normal nasal mucosa was absent. We also found P100 Protein was also expressed in epithelial layer and gland of nasal polyps, but it was missing in 80% of eosinophils.
The second part: we confirm that IL-18 is expressed in the epithelial cell line A549, and the expression increases under the LPS stimulation. The mature IL-18 is only expressed in the A549 cells stimulated by LPS, but the normal A549 is absent. We also found that IL-4 can weaken the IL-18 expression in the A549 of the epithelial cell line. The expression of cytokines in the epithelial cells can also be regulated by each other.
The third part: the mechanism of P100 protein involvement in inflammation may be involved in the IL-4/STAT6 signal transduction pathway, but not involved in the process of cytokine production induced by the LPS/TLR4 signaling pathway. In addition, the P100 protein may be closely related to the cell cycle, and its participation in cell cycle may not pass through the c-Myc pathway, but through other signaling pathways.
Conclusion: the expression of.IL-18 in nasal polyps is increased in IL-18, normal nasal mucosa and nasal polyps, and the expression of mature IL-18 is only in nasal polyps. The absence of.IL-18 in normal nasal mucosa may play an important role in the formation of nasal polyps. Nasal epithelial cells can secrete a large number of thin cells under the stimulation of inflammatory factors. Cell factors such as IL-18, IL-4, IFN-r, and so on, nasal epithelial cells are the first barrier to contact antigen, may be the '''initiator' in the occurrence and development of nasal polyps. The P100 protein participates in the inflammatory mechanism of nasal polyps through the IL-4/STAT6 signal transduction pathway. The P100 protein may be closely related to the cell cycle, and is involved in the proliferation of nasal polyps and in the nose. Polyps play a very important role in the formation of polyps.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R392.1;R765.25

【参考文献】