【摘要】：盡管近年來(lái)對(duì)慢性鼻竇炎伴鼻息肉的發(fā)病機(jī)制和分子機(jī)制方面取得重大研究進(jìn)展,但是關(guān)于慢性鼻竇炎伴鼻息肉中復(fù)雜的炎癥網(wǎng)絡(luò)以及對(duì)其調(diào)控仍認(rèn)識(shí)的不充分。越來(lái)越多研究顯示Sirtuins家族成員(Sirt1-Sirt7 )在各種急性或慢性炎性疾病,免疫疾病和代謝性疾病的病理進(jìn)展的衰老,炎癥,細(xì)胞死亡和氧化應(yīng)激過(guò)程中發(fā)揮保護(hù)作用。前期的研究表明Sirt1和Sirt6參與脂多糖誘導(dǎo)鼻黏膜上皮細(xì)胞核表達(dá)的HMGB1易位和主動(dòng)釋放。然而,Sirtuins在鼻息肉中的作用和分子機(jī)制尚不清楚,是否調(diào)控慢性鼻竇炎伴鼻息肉中各種促炎細(xì)胞因子(包括IL-1β,IL-5,IL-8, IL-10,IL-17和HMGB1)和活性氧(ROS)。因此,Sirtuins在鼻息肉中是否存在與炎癥和氧化應(yīng)激相關(guān)的特定分子機(jī)制和其調(diào)節(jié)機(jī)制需要深入研究。目的:觀察Sirt1-Sirt6在慢性鼻竇炎伴鼻息肉與正常鼻黏膜組織中的表達(dá)差異,以及探討Sirt1在鼻黏膜上皮細(xì)胞炎癥反應(yīng)和氧化應(yīng)激中相關(guān)的分子作用機(jī)制。方法:收集52例鼻息肉組織及15例正常的中鼻甲組織作為對(duì)照。通過(guò)實(shí)時(shí)熒光定量PCR,免疫印跡和免疫組織化學(xué)方法檢測(cè)Sirtuins,炎癥細(xì)胞因子(IL-1β, IL5,IL-8, IL-10, IL-17和HMGB1等),氧化應(yīng)激相關(guān)抗氧化酶GPX4的表達(dá)水平。用LPS刺激鼻息肉的原發(fā)性人鼻腔上皮(HNE)細(xì)胞。用基因克隆技術(shù)過(guò)表達(dá)HNE細(xì)胞中的Sirt1表達(dá)水平,用siRNA技術(shù)敲低HNE細(xì)胞中的Sirt1表達(dá)。通過(guò)MTT檢測(cè)敲低和過(guò)表達(dá)的Sirt1對(duì)HNE增殖的影響。通過(guò)流式細(xì)胞術(shù)(FACS)觀察HNE細(xì)胞的分化。運(yùn)用FACS技術(shù),通過(guò)膜聯(lián)蛋白V/7AAD染色測(cè)定細(xì)胞死亡,通過(guò)BODIPY染色評(píng)估ROS產(chǎn)生。結(jié)果:在本研究中,我們發(fā)現(xiàn)Sirt1, Sirt3和Sirt6 mRNA和蛋白表達(dá)水平在CRSwNP組織中比正常鼻粘膜組織中低。在CRSwNP中HMGB1, Nrf2, NF-kB和pIkB的蛋白水平增加。然而,GPX4和p-STAT在CRSwNP中表達(dá)降低。此外,在鼻息肉中GPX4mRNA表達(dá)與IL-1β,IL-5, IL-8和IL-17mRNA表達(dá)呈負(fù)相關(guān)。在體外細(xì)胞實(shí)驗(yàn)中,我們建立了 Sirt1過(guò)表達(dá)的人鼻黏膜上皮(HNE )細(xì)胞,并通過(guò)RNA干擾技術(shù)敲低Sirt1。我們發(fā)現(xiàn)Sirt1敲低后明顯抑制HNE細(xì)胞生長(zhǎng),并誘導(dǎo)細(xì)胞死亡。并且Sirt1敲低能影響HNE細(xì)胞的纖毛分化,以及HNE細(xì)胞中的CK14表達(dá)水平增加。Sirt1通過(guò)調(diào)控GPX4, NF-kB和STAT通路來(lái)調(diào)節(jié)HNE細(xì)胞的氧化應(yīng)激能力。此外,在LPS刺激下Sirt1敲低的HNE細(xì)胞發(fā)生ROS積累和ferroptosis細(xì)胞死亡。結(jié)論:這些研究結(jié)果揭示了 Sirtuins在鼻息肉中的表達(dá)異常。Sirt1在鼻黏膜上皮細(xì)胞的生長(zhǎng)、分化、和炎癥氧化應(yīng)激反應(yīng)中扮演了重要角色,提供了對(duì)免疫應(yīng)答的表觀遺傳調(diào)節(jié)機(jī)制的了解。因此,它可以作為控制和預(yù)防鼻息肉潛在的治療靶點(diǎn)。
[Abstract]:Although great progress has been made in the pathogenesis and molecular mechanism of chronic sinusitis and nasal polyps in recent years, the complex inflammatory network and its regulation in chronic sinusitis and nasal polyps are still not well understood. More and more studies have shown that Sirtuins family members (Sirt1-Sirt7) play a protective role in the progression of aging, inflammation, cell death and oxidative stress in various acute or chronic inflammatory diseases, immune diseases and metabolic diseases. Previous studies have shown that Sirt1 and Sirt6 are involved in HMGB1 translocation and active release induced by lipopolysaccharide. However, the role and molecular mechanism of Sirtuins in nasal polyps are unclear, and whether the regulation of various pro-inflammatory cytokines (including IL-1 尾, IL-5,IL-8, IL-10,IL-17 and HMGB1) and reactive oxygen (ROS). (Ros) in chronic sinusitis and nasal polyps is unclear. Therefore, the existence of specific molecular mechanisms and regulatory mechanisms related to inflammation and oxidative stress in nasal polyps requires further study. Aim: to investigate the expression of Sirt1-Sirt6 in chronic sinusitis with nasal polyps and normal nasal mucosa, and to explore the molecular mechanism of Sirt1 in the inflammatory response of nasal epithelial cells and oxidative stress. Methods: 52 cases of nasal polyp and 15 cases of normal middle turbinate were collected as control group. The expression of Sirtuins, inflammatory cytokines (IL-1 尾, IL5,IL-8, IL-10, IL-17, HMGB1, etc.) and the expression of oxidative stress-related antioxidant enzymes (GPX4) were detected by real-time quantitative PCR, blotting and immunohistochemistry. Primary human nasal epithelial (HNE) cells of nasal polyps were stimulated by LPS. The expression level of Sirt1 in HNE cells was overexpressed by gene cloning technique, and Sirt1 expression in HNE cells was reduced by siRNA technique. The effect of knockout and overexpression of Sirt1 on HNE proliferation was detected by MTT. The differentiation of HNE cells was observed by flow cytometry (FACS). FACS technique was used to detect cell death by V/7AAD staining and ROS production was evaluated by BODIPY staining. Results: in this study, we found that the expression levels of Sirt1, Sirt3 and Sirt6 mRNA and protein were lower in CRSwNP than in normal nasal mucosa. The protein levels of HMGB1, Nrf2, NF-kB and pIkB increased in CRSwNP. However, the expression of GPX4 and p-STAT decreased in CRSwNP. In addition, the expression of GPX4mRNA was negatively correlated with the expression of IL-1 尾, IL-5, IL-8 and IL-17mRNA in nasal polyps. In vitro, we established human nasal epithelial (HNE) cells with overexpression of Sirt1, and knocked down Sirt1. by RNA interference technique. We found that Sirt1 knockout significantly inhibited the growth of HNE cells and induced cell death. Furthermore, Sirt1 knockout can affect the ciliated differentiation of HNE cells and increase the expression of CK14 in HNE cells. Sirt1 regulates the oxidative stress ability of HNE cells by regulating GPX4, NF-kB and STAT pathways. In addition, ROS accumulation and ferroptosis cell death occurred in HNE cells with low Sirt1 knockout stimulated by LPS. Conclusion: these results reveal the abnormal expression of Sirtuins in nasal polyps. Sirt1 plays an important role in the growth, differentiation, and inflammatory oxidative stress of nasal epithelial cells. It provides an understanding of the epigenetic regulation mechanism of immune response. Therefore, it can be used as a potential therapeutic target for the control and prevention of nasal polyps.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R765

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相關(guān)碩士學(xué)位論文 前2條

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