本文選題：慢性鼻-鼻竇炎 + 樹突狀細(xì)胞�。� 參考：《重慶醫(yī)科大學(xué)》2016年博士論文

【摘要】：慢性鼻鼻竇炎(chronic rhinosinusitis,CRS)是一類以鼻腔和鼻竇粘膜的慢性炎癥為特征的耳鼻咽喉頭頸外科常見多發(fā)病,目前學(xué)術(shù)界普遍將CRS分為慢性鼻鼻竇炎伴息肉(CRSw NP)和慢性鼻鼻竇炎不伴息肉(CRSs NP)兩個(gè)疾病亞型。CRS的治療目的也僅僅是控制臨床癥狀和減少并發(fā)癥的發(fā)生,目前還不能達(dá)到根治的目的,原因在于CRS的發(fā)病機(jī)制復(fù)雜,尚不完全清楚。最近的研究表明,可能與遺傳基因、環(huán)境、變態(tài)反應(yīng)、CRS的重塑、解剖結(jié)構(gòu)、先天性免疫、細(xì)菌生物膜等有關(guān),然而究竟是哪種因素是引起CRS的關(guān)鍵因素,有必要進(jìn)行深入的研究。但目前研究顯示,樹突狀細(xì)胞(dendritic cell,DC)、Th17/Treg失衡和micro RNAs(mi RNAs)是CRS發(fā)病的重要基礎(chǔ),然而其的發(fā)生機(jī)制尚未闡明,本研究將從DC-Th軸探討mi RNA在CRS中的作用。DC是機(jī)體內(nèi)一種專職抗原提呈細(xì)胞,因其表面有樹突狀突起而得名。DC在免疫系統(tǒng)中發(fā)揮極其重要的作用,不同的DC可以相互作用和調(diào)節(jié)。DC是如何影響免疫系統(tǒng)中其它細(xì)胞的功能呢?一方面,DC對外來微生物進(jìn)行識別、攝取、加工處理和提呈,在天然免疫中發(fā)揮抗微生物感染的作用;另一方面,DC捕獲抗原后能與初始T(na?ve T)細(xì)胞相互作用,激活na?ve T細(xì)胞,并通過分泌IL-17、IL-12、IL-10等細(xì)胞因子調(diào)控輔助性T(Th)細(xì)胞的誘導(dǎo)、分化,從而對啟動(dòng)和維系Th細(xì)胞反應(yīng)起到?jīng)Q定性作用,調(diào)節(jié)獲得性免疫應(yīng)答。DC作為抗原遞呈細(xì)胞,功能強(qiáng)大,能夠阻止微環(huán)境中病原體的侵入,成熟的DC可以遷移到淋巴結(jié)而發(fā)生抗原遞呈的作用,還可刺激T細(xì)胞增殖。在T細(xì)胞介導(dǎo)的機(jī)體細(xì)胞免疫中,CD4+Th細(xì)胞具有重要意義,na?ve T細(xì)胞在抗原提呈細(xì)胞的誘導(dǎo)下可分化為Th1細(xì)胞、Th2細(xì)胞、Th17細(xì)胞和T調(diào)節(jié)性細(xì)胞(T regulatory cells,Treg)發(fā)揮不同作用。Mi RNAs是一類單鏈的非編碼RNA分子,長約19-22核苷酸,能夠干預(yù)靶基因沉默的轉(zhuǎn)錄后調(diào)控和抑制目標(biāo)m RNA翻譯蛋白質(zhì)。因此,mi RNAs是一類有望調(diào)節(jié)上氣道炎癥的微小分子,也就是在CRS中同樣發(fā)揮作用。借助以上關(guān)于mi RNAs在各慢性炎癥性疾病中作用以及mi RNA在DC中作用的研究結(jié)果,我們推測在CRS發(fā)病機(jī)制中DC-Th軸的異常必然有相當(dāng)數(shù)量的mi RNA參與其中。然而,參與DC-Th軸異常的mi RNA表達(dá)譜具體如何?不同亞類的CRS是否存在共有和特異的差異表達(dá)mi RNA?mi RNA對DC-Th軸調(diào)控的機(jī)制如何?是否可以通過干預(yù)差異表達(dá)mi RNA調(diào)控DC-Th軸的平衡,最終達(dá)到疾病預(yù)防和治療的目的?為了更有效的治療CRS,有必要探索以上科學(xué)問題。本研究以CRS患者為研究對象,以DC-Th軸為主線,以mi RNA對DC的調(diào)控為切入點(diǎn),明確CRS患者DC相關(guān)的mi RNA表達(dá)譜,探討mi RNA對DC-Th軸的調(diào)控作用,以期從DC層面闡明CRS中Th細(xì)胞失衡的上游分子事件及調(diào)控機(jī)制,為CRS發(fā)病機(jī)制的研究及臨床防治策略的建立提供新的靶點(diǎn)和新的思路。第一部分不同類型CRS患者外周血中DC表型分布的研究目的:采用流式細(xì)胞術(shù)(flow cytometry,FCM)檢測DC在CRS外周血中的表型、數(shù)量、分布特征,比較它們之間的差異,觀察DC在CRS發(fā)病機(jī)制中的作用。方法:收集2014年7月-2015年8月67例CRS患者,參照EPOS2012的診斷標(biāo)準(zhǔn)分為4個(gè)組:(1)正常對照組:選擇單純鼻中隔偏曲患者(13例);(2)CRSs NP:為不伴鼻息肉的CRS患者(18例);(3)atopic CRSw NP:伴變應(yīng)性體質(zhì)的慢性鼻-鼻竇炎伴鼻息肉患者(18例);(4)non-atopic CRSw NP:不伴變應(yīng)性體質(zhì)的慢性鼻-鼻竇炎伴鼻息肉患者(18例)。采用淋巴細(xì)胞分離液密度梯度離心法獲得外周血單個(gè)核細(xì)胞(peripheral blood mononuclear cells,PBMC),再用免疫磁珠法分離得到CD+14單核細(xì)胞,在白細(xì)胞介素-4(interleukin-4,IL-4)和粒細(xì)胞集落刺激生物因子(granulocyte-macrophage colony stimulating Factor,GM-CSF)誘導(dǎo)培養(yǎng)后得到DCs,通過流式細(xì)胞術(shù),檢測CRS外周CD80、CD83、CD1a和CD86(DC成熟和活化標(biāo)志)的表達(dá)。評價(jià)不同類型CRS外周血DC的表型、數(shù)量及分布特征。結(jié)果:CRS患者外周血中DC的分布情況:從FCM散點(diǎn)圖可以看出成熟DCs的陽性比例:control 38.98%;CRSs NP 57.77%;atopic CRSw NP 84.87%;non-atopic CRSw NP 82.94%。同對照組相比,CRS中成熟DCs均高于對照組,而息肉組中成熟DCs高于非息肉組,伴有變應(yīng)性體質(zhì)的明顯高于不伴有變應(yīng)性體質(zhì)。P0.05,差異有統(tǒng)計(jì)學(xué)意義。結(jié)論:DCs在CRS外周血中增多,DC在CRS患者的發(fā)病機(jī)制中可能扮演著重要的角色;而atopic組表現(xiàn)為更多的DC分布,提示變應(yīng)性因素可能促使DC增多。因此,對于伴有變應(yīng)性體質(zhì)的CRS患者,除了手術(shù)治療外,還應(yīng)積極抗過敏治療。第二部分確定CRS患者外周血DC的mi RNA表達(dá)譜目的:通過對CRS患者樣本外周血中DC提取RNA后進(jìn)行基因芯片檢測,再用實(shí)時(shí)熒光定量PCR技術(shù)進(jìn)一步驗(yàn)證芯片結(jié)果,確定出CRS患者外周血中DC的mi RNA表達(dá)譜,并進(jìn)行定量分析,篩選差異表達(dá)mi RNA。評價(jià)CRS患者不同亞型CRS共有和特有的差異表達(dá)mi RNA。旨在探討篩選出具有代表性的差異表達(dá)mi RNA在CRS發(fā)生發(fā)展中的作用。方法:分別收集3個(gè)組的CRS患者及對照組樣本的外周血,先分離PBMC,予以免疫磁珠法分離CD14+的單核細(xì)胞,予以脂多糖(Lipopolysaccharide,LPS)誘導(dǎo)為成熟的DCs;再利用Trizol裂解蛋白之后,酚-氯仿反復(fù)抽提,提取DC中總RNA,采用mi RNA基因芯片分析技術(shù)進(jìn)行檢測,再用實(shí)時(shí)熒光定量PCR技術(shù)進(jìn)一步驗(yàn)證芯片結(jié)果,確定出CRS患者外周血中DC的mi RNA表達(dá)譜,并進(jìn)行定量分析,篩選差異表達(dá)mi RNA。結(jié)果:Mi RNA基因芯片結(jié)果顯示:同對照組相比,不同類型的8CRS患者外周血中DCs有不同的差異表達(dá)的mi RNA,在這3種類型的CRS中共有31種差異表達(dá)的mi RNAs,其中有5種mi RNAs表達(dá)是上調(diào),有25種mi RNAs表達(dá)是下調(diào),而mi RNA-1290表達(dá)在CRSs NP中是下調(diào),在atopic CRSw NP和non-atopic CRSw NP中是上調(diào)。結(jié)論:通過對3種類型的CRSmi RNA基因表達(dá)的比較,我們推測差異表達(dá)的mi RNA有可能通過調(diào)節(jié)DCs來干預(yù)CRS的發(fā)病機(jī)制,這些mi RNAs有可能是治療CRS的一個(gè)新靶點(diǎn)。第三部分從DC-Th軸探討mi R-150-5P在CRS中的作用目的:通過DC與原始T細(xì)胞(na?ve T細(xì)胞)共培養(yǎng)后,利用FCM檢測T細(xì)胞的增殖情況和培養(yǎng)后上清液的IL-17的濃度情況,從DC-Th軸探討mi R-150-5P在CRS中的表達(dá),評價(jià)在CRS中mi R-150-5P引起Th17/Treg失衡的上游事件和它的免疫調(diào)節(jié)因素。方法:從不同類型CRS外周血中分離的DCs,誘導(dǎo)成熟后,轉(zhuǎn)染mi R-150-5P模擬劑/抑制劑(mimic/inhibitor)后,與CFSE標(biāo)記的同源異體的na?ve T細(xì)胞共培養(yǎng)5天(1:20),收集T細(xì)胞,予anti-CD3和anti-CD28刺激,通過FCM檢測CFSE標(biāo)記的T細(xì)胞比率,通過酶聯(lián)免疫吸附試驗(yàn)(enzyme-linked immuno sorbent assay,ELISA)檢測IL-17的濃度。結(jié)果:同未轉(zhuǎn)染相比,轉(zhuǎn)染mi R-150-5P mimic組的T細(xì)胞增殖和Il-17增加,而轉(zhuǎn)染mi R-150-5P inhibitor組下降,有DCs共培養(yǎng)組高于未共培養(yǎng)組,CRS患者組高于正常對照組,P0.05,有統(tǒng)計(jì)學(xué)意義。結(jié)論:mi R-150-5P在CRS中的表達(dá)是上調(diào)表達(dá),mi R-150-5P mimic促進(jìn)T細(xì)胞增殖和IL-17分泌,mi R-150-5P inhibitor抑制T細(xì)胞增殖和IL-17分泌。mi R-150-5P通過DC-Th軸影響CRS發(fā)病的。第四部分預(yù)測并驗(yàn)證CRS患者DC中mi R-150-5P可能作用的靶基因目的:探討差異表達(dá)mi R-150-5P可能作用的靶基因及靶蛋白,以期探討通過干預(yù)差異表達(dá)mi R-150-5P對CRS疾病預(yù)防和治療的意義。方法:采用生物信息學(xué)分析軟件Target Scan、Pic Tar、Mi Randa和mi RBase Targets對與DC分化、成熟和功能相關(guān)的mi R-150-5P靶基因進(jìn)行靶點(diǎn)預(yù)測,通過蛋白質(zhì)免疫印跡試驗(yàn)(Western blot,WB)驗(yàn)證靶蛋白,通過熒光素酶報(bào)告試驗(yàn),將靶蛋白3’UTR區(qū)域克隆至含蟲熒光素酶報(bào)告基因3’UTR區(qū),同時(shí)合成mi R-150-5P片段將二者共轉(zhuǎn)染HEK293細(xì)胞(1x104),孵育48h后,對熒光素酶活性進(jìn)行檢測,驗(yàn)證該靶基因是否為mi R-150-5P的靶基因。結(jié)果:通過生物學(xué)軟件預(yù)測早期生長反應(yīng)蛋白2(early growth response 2,EGR2)mi R-150-5p的靶基因,并通過WB試驗(yàn)和熒光素酶報(bào)告試驗(yàn)驗(yàn)證EGR2是mi R-150-5P的靶基因。結(jié)論:Mi R-150-5p和它的靶基因EGR2在CRS的發(fā)病機(jī)制中起了重要的作用。
[Abstract]:Chronic rhinosinusitis (CRS) is a common type of Otolaryngology and head and neck surgery characterized by chronic inflammation of the nasal cavity and sinus mucosa. At present, CRS is widely divided into two subtypes of chronic rhinosinusitis with polyps (CRSw NP) and chronic rhinosinusitis without polyps (CRSs NP). Only to control clinical symptoms and reduce the occurrence of complications is not yet to be achieved, because the pathogenesis of CRS is complex and not completely clear. Recent studies have shown that it may be related to genetic genes, environment, allergy, remolding of CRS, anatomical structure, innate immunity, bacterial biofilm and so on. But what is it Seed factors are the key factors that cause CRS, which is necessary for in-depth study. However, the current research shows that dendritic cell (DC), Th17/Treg imbalance and micro RNAs (MI RNAs) are the important basis for CRS. However, the mechanism of the occurrence of CRS has not been elucidated. The specific antigen presenting cells, named.DC, whose surface has a dendritic protuberance, play an important role in the immune system. Different DC can interact and regulate how.DC affects the function of other cells in the immune system. On the one hand, DC can identify, absorb, processing and presenting foreign microorganisms in natural immunity. On the other hand, the DC capture antigen can interact with the initial T (Na? Ve T) cells to activate na? Ve T cells and regulate the induction and differentiation of the auxiliary T cells by secreting IL-17, IL-12, IL-10 and other cytokines, which play a decisive role in initiating and maintaining the cell response and regulating acquired immunity. Responding to.DC as an antigen presenting cell, it has strong function and can prevent the invasion of pathogens in microenvironment. Mature DC can migrate to lymph nodes and produce antigen presentation, and also stimulate the proliferation of T cells. In the cellular immunity mediated by T cells, CD4+Th cells are of great importance, and na? Ve T cells are induced by antigen presenting cells. Th1 cells, Th2 cells, Th17 cells and T regulatory cells (T regulatory cells, Treg) play different roles..Mi RNAs is a class of single strand non coded RNA molecules, with a length of about 19-22 nucleotides, which can interfere with the post transcriptional regulation of target gene silence and inhibit the target m to translate the protein. Small molecules of inflammation are also played in CRS. With the help of the above research on the role of MI RNAs in various chronic inflammatory diseases and the role of MI RNA in DC, we speculate that a considerable amount of MI RNA is bound to be involved in the abnormalities of the DC-Th axis in the pathogenesis of CRS. How is the spectrum specific? Whether there is a common and specific differential expression of the different subclasses of CRS expression of the MI RNA? Mi RNA mechanism for the regulation of the DC-Th axis? Is it possible to regulate the equilibrium of the DC-Th axis by interfering with the differential expression of MI RNA and ultimately to achieve the purpose of disease prevention and treatment? For the more effective treatment of CRS, it is necessary to explore the above scientific problems. Taking the CRS patients as the research object, taking the DC-Th axis as the main line and taking the regulation of the DC as the breakthrough point of the MI RNA, the DC related mi RNA expression profiles of the CRS patients are clarified, and the regulation effect of MI RNA on the DC-Th axis is discussed in order to clarify the upstream molecular events and the control mechanism of the imbalance of the MI RNA cell, so as to study the pathogenesis and the clinical prevention strategy. To provide new targets and new ideas. Study the DC phenotypic distribution in peripheral blood of different types of CRS patients in the first part: use flow cytometry (flow cytometry, FCM) to detect the phenotype, quantity and distribution of DC in the peripheral blood of CRS, compare the difference between them, and observe the role of DC in the pathogenesis of CRS. Methods: collect 2014 In July -2015 August, 67 cases of CRS patients were divided into 4 groups according to the diagnostic criteria of EPOS2012: (1) the normal control group (13 cases); (2) CRSs NP: as CRS patients without nasal polyps (18); (3) atopic CRSw NP: associated with allergic constitution of chronic rhinosinusitis with nasal polyps (18 cases); (4) non-atopic CRSw NP: Chronic rhinosinusitis with nasal polyps (18 cases) with allergic constitution (18 cases). Peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMC) were obtained by density gradient centrifugation by lymphocyte separation solution, and CD+14 mononuclear cells were separated by immunomagnetic beads, and interleukin -4 (interleukin-4, IL-4) and granulocyte colony stimulation were obtained. Granulocyte-macrophage colony stimulating Factor (GM-CSF) was induced and cultured to obtain DCs. The expression of CRS peripheral CD80, CD83, CD1a and CD86 (DC maturity and activation markers) was detected by flow cytometry. The phenotypes, quantity and distribution characteristics of different types of peripheral blood were evaluated. The positive proportion of mature DCs can be seen from the FCM scatter plot: control 38.98%, CRSs NP 57.77%, atopic CRSw NP 84.87%, non-atopic CRSw NP 82.94%. is higher than the control group, and the mature polyps in the polyp group are higher than those of non polyps, and the allergic constitution is obviously higher than that of the non allergic constitution. Statistical significance. Conclusion: DCs in the peripheral blood of CRS increased, DC in the pathogenesis of CRS patients may play an important role, and the atopic group is more DC distribution, suggesting that the allergic factors may promote the increase of DC. Therefore, for CRS patients with allergic constitution, besides surgical treatment, should also be active anti allergic treatment. The two part was to determine the MI RNA expression profile of DC in peripheral blood of CRS patients. By gene chip detection after extracting RNA in the peripheral blood of CRS patients, the results were further verified by real-time fluorescent quantitative PCR technique, and the MI RNA expression spectrum of DC in the peripheral blood of the patients was determined, and the quantitative analysis was carried out to screen the differential expression. CRS common and specific differential expression of MI RNA. in different subtypes of CRS patients was designed to explore the role of a representative differential expression of MI RNA in the development of CRS. Methods: the peripheral blood of 3 groups of CRS patients and the control group were collected, the PBMC was isolated and the mononuclear cells of CD14+ were isolated by immunomagnetic beads, and the lipopolysaccharide was given. Lipopolysaccharide, LPS) was induced to be a mature DCs. After using Trizol cracking protein, phenol chloroform was repeatedly extracted to extract the total RNA in DC. Mi RNA gene chip analysis technique was used to detect the total RNA. The results were further verified by real-time fluorescent quantitative PCR technique, and the DC Mi expression profiles in the peripheral blood of the CRS patients were determined and quantified. Analysis of the results of differential expression of MI RNA.: Mi RNA gene chip results showed that compared with the control group, DCs had different mi RNA in the peripheral blood of different types of 8CRS patients, and there were 31 differentially expressed mi RNAs in these 3 types of CRS, of which 5 kinds of MI tables were up regulation, and 25 kinds of expressions were down regulated. The 0 expression is down down in CRSs NP and up up in atopic CRSw NP and non-atopic CRSw NP. Conclusion: by comparing the expression of the 3 types of CRSmi RNA gene, we speculate that the differential expression of MI RNA may interfere with the pathogenesis by regulating them. Objective: To investigate the role of MI R-150-5P in CRS. After co culture of DC and primitive T cells (Na ve T cells), the expression of T cells in T cells and the IL-17 concentration of the supernatant were detected by FCM. Methods: DCs, isolated from the peripheral blood of different types of CRS, was induced to mature, after transfection of MI R-150-5P analogue / inhibitor (mimic/inhibitor), and the CFSE labeled na? Ve T cells were co cultured for 5 days (1:20), and T cells were collected. The concentration of IL-17 was detected by enzyme-linked immuno sorbent assay (ELISA). Results: the proliferation and Il-17 increase of T cells in the MI R-150-5P mimic group were compared with those in the untransfected mi R-150-5P mimic group. Conclusion: the expression of MI R-150-5P in CRS is up to up expression, and MI R-150-5P mimic promotes the proliferation of T cells and the secretion of IL-17. Mi R-150-5P inhibitor inhibits the proliferation and secretion of T cells. To discuss the potential target genes and target proteins that differentially express mi R-150-5P, in order to explore the significance of differentially expressed mi R-150-5P for the prevention and treatment of CRS disease. Methods: the bioinformatics analysis software Target Scan, Pic Tar, Mi Randa, and Mi fragments are used for the differentiation, maturation and function related target genes. Target protein test (Western blot, WB) was used to test the target protein. The target protein 3 'UTR region was cloned to the 3' UTR region of the luciferase reporter gene, and the MI R-150-5P fragment was synthesized by the luciferase reporter assay, and the two groups were co transfected to HEK293 cell (1x104), and the luciferase activity was detected after incubating 48h. To verify whether the target gene is a target gene for MI R-150-5P. Results: the target gene of the early growth reaction protein 2 (early growth response 2, EGR2) mi R-150-5p was predicted by biological software, and the target gene of MI R-150-5P was verified by WB test and Luciferase Report test. The mechanism of disease plays an important role.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R765

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