本文選題：Il-18 + p100　； 參考：《天津醫(yī)科大學(xué)》2011年博士論文

【摘要】：目的：鼻息肉是鼻腔外側(cè)壁粘膜突入鼻腔形成的新生物,發(fā)病機(jī)制不明,其本質(zhì)是粘膜的慢性持續(xù)性炎癥。白細(xì)胞介素18(IL-18)是近年來發(fā)現(xiàn)的前炎性細(xì)胞因子,在感染、炎癥、自身免疫性疾病、組織的炎性損傷等過程中發(fā)揮非常重要的調(diào)節(jié)作用。p100蛋白是一種多功能蛋白,它可與多種蛋白轉(zhuǎn)錄因子結(jié)合,廣泛的存在于各種組織細(xì)胞。然而,查閱文獻(xiàn),未見IL-18和p100蛋白在鼻息肉中的表達(dá)情況的報(bào)道。由此,我們提出疑問：IL-18和p100在鼻息肉中的表達(dá)情況如何呢?二者與鼻息肉的發(fā)病是否有關(guān)系呢?二者在鼻息肉的發(fā)病機(jī)制中到底扮演什么樣的角色呢?其深入的分子機(jī)制又是如何?此課題我們檢測(cè)IL-18和p100在鼻息肉中的表達(dá)情況,并進(jìn)一步探討二者在鼻息肉形成過程中可能的作用機(jī)制。 方法： 第一部分：IL-18等細(xì)胞因子及p100蛋白在鼻息肉中的表達(dá)與定位。收集鼻息肉和正常鼻粘膜臨床標(biāo)本,將其分為兩部分,一部分制作石蠟病理切片,進(jìn)行HE及免疫組化染色,觀察鼻息肉炎細(xì)胞構(gòu)成及浸潤(rùn)情況,觀察IL-18、p100蛋白在鼻息肉中的表達(dá)情況。另一部分液氮儲(chǔ)存,勻漿并變性蛋白后進(jìn)行Western Blotting檢測(cè),在蛋白水平進(jìn)一步的驗(yàn)證IL-18、p100蛋白在鼻息肉中的表達(dá)情況。 第二部分：IL-18在鼻息肉形成過程中可能作用機(jī)制的研究。這部分實(shí)驗(yàn)中,我們以A549細(xì)胞系作為呼吸道上皮細(xì)胞的代表,以炎癥因子LPS,細(xì)胞因子IL-4刺激A549細(xì)胞,觀察IL-18的表達(dá)情況以明確上皮細(xì)胞在炎癥狀態(tài)下是否會(huì)分泌IL-18等細(xì)胞因子以及上皮細(xì)胞分泌的細(xì)胞因子之間是否會(huì)相互調(diào)控,以進(jìn)一步的證明IL-18參與了鼻息肉形成的病理機(jī)制,且上皮細(xì)胞可能是鼻息肉病理機(jī)制發(fā)生的"initiator"。 第三部分：p100在鼻息肉形成過程中可能作用機(jī)制的。在這部分實(shí)驗(yàn)中,我們將實(shí)驗(yàn)分成兩個(gè)部分,(1)p100是否參與IL-4/STAT6和LPS/TLR4信號(hào)通路的研究；(2)p100是否參與增殖和細(xì)胞周期的研究。在第一部分實(shí)驗(yàn)中我們的技術(shù)路線是：在細(xì)胞水平,以LPS, IL-4刺激A549細(xì)胞,觀察IL-8, IL-6等細(xì)胞因子,STAT6, p100的表達(dá)變化情況。在動(dòng)物水平,我們以O(shè)VA誘導(dǎo)小鼠哮喘模型以模擬IL-4/STAT6信號(hào)通路,以LPS誘導(dǎo)急性肺炎模型模擬LPS/TLR4信號(hào)通路,取小鼠肺組織,觀察支氣管上皮細(xì)胞p100表達(dá)情況。在第二部分實(shí)驗(yàn)中我們又分為兩個(gè)小的實(shí)驗(yàn)①p100與增殖狀態(tài)關(guān)系的初步研究和②p100與細(xì)胞周期相關(guān)轉(zhuǎn)錄因子c-Myc的關(guān)系的研究,實(shí)驗(yàn)①的技術(shù)路線是取小鼠不同增殖狀態(tài)的組織和不同增殖狀態(tài)的外周血細(xì)胞檢測(cè)p100蛋白的表達(dá)水平。實(shí)驗(yàn)②的技術(shù)路線是第一,我們培養(yǎng)裂解hela細(xì)胞,分別以p100抗體釣取c-Myc蛋白,以c-Myc抗體釣取p100蛋白,WB檢驗(yàn)釣取結(jié)果,以明確p100蛋白是否與c-Myc蛋白相結(jié)合。第二,以不同的質(zhì)粒轉(zhuǎn)染hela細(xì)胞,建造p100過表達(dá),和p100抑制的細(xì)胞模型,檢測(cè)c-Myc的表達(dá)情況,以視p100表達(dá)是否對(duì)c-Myc蛋白表達(dá)產(chǎn)生影響。 結(jié)果： 第一部分：我們發(fā)現(xiàn)IL-18、 IL-4、 IFN-y在正常鼻粘膜及鼻息肉的上皮層、腺體及間質(zhì)的炎性細(xì)胞中均有表達(dá),且在鼻息肉中表達(dá)增加,在嗜酸粒鼻息肉中表達(dá)增加更明顯,成熟型IL-18僅在鼻息肉中表達(dá),而正常鼻粘膜中缺如。我們還發(fā)現(xiàn)p100蛋白在鼻息肉上皮層和腺體中也大量表達(dá),但在80%的嗜酸粒細(xì)胞中表達(dá)缺失。 第二部分：我們證實(shí)IL-18在上皮細(xì)胞系A(chǔ)549中表達(dá),且在LPS刺激下表達(dá)增加,成熟型IL-18僅在LPS刺激的A549細(xì)胞中表達(dá),而正常A549中缺如。我們還發(fā)現(xiàn)IL-4能減弱IL-18在上皮細(xì)胞系A(chǔ)549中表達(dá)。細(xì)胞因子在上皮細(xì)胞的表達(dá)也可相互調(diào)控。 第三部分：p100蛋白參與炎癥的機(jī)制可能是參與了IL-4/STAT6信號(hào)轉(zhuǎn)導(dǎo)通路,而沒有參與LPS/TLR4信號(hào)通路誘導(dǎo)的細(xì)胞因子生成過程。另外,p100蛋白可能與細(xì)胞周期密切相關(guān),其參與細(xì)胞周期可能不是通過c-Myc途徑,而是通過其它的信號(hào)通路。 結(jié)論：IL-18、p100正常鼻粘膜及鼻息肉中均大量表達(dá)。IL-18在鼻息肉中表達(dá)增加,成熟型IL-18僅在鼻息肉中表達(dá),而正常鼻粘膜中缺如。IL-18在鼻息肉的形成過程中可能發(fā)揮重要的作用。鼻粘膜上皮細(xì)胞可在致炎因素的刺激下分泌大量的細(xì)胞因子如IL-18、 IL-4、IFN-r等,鼻上皮細(xì)胞是接觸抗原的第一道屏障,可能是鼻息肉等疾病發(fā)生發(fā)展的‘''initiator"。 p100蛋白通過IL-4/STAT6信號(hào)轉(zhuǎn)導(dǎo)通路參與了鼻息肉的炎癥機(jī)制,p100蛋白可能與細(xì)胞周期密切相關(guān),參與了鼻息肉的增生,在鼻息肉的形成過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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R392.1;R765.25

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