从DC-TH轴探讨miR-150-5P在慢性鼻—鼻窦炎中的作用

发布时间：2018-05-03 02:23

本文选题：慢性鼻-鼻窦炎 + 树突状细胞　；参考：《重庆医科大学》2016年博士论文

【摘要】：慢性鼻鼻窦炎(chronic rhinosinusitis,CRS)是一类以鼻腔和鼻窦粘膜的慢性炎症为特征的耳鼻咽喉头颈外科常见多发病,目前学术界普遍将CRS分为慢性鼻鼻窦炎伴息肉(CRSw NP)和慢性鼻鼻窦炎不伴息肉(CRSs NP)两个疾病亚型。CRS的治疗目的也仅仅是控制临床症状和减少并发症的发生,目前还不能达到根治的目的,原因在于CRS的发病机制复杂,尚不完全清楚。最近的研究表明,可能与遗传基因、环境、变态反应、CRS的重塑、解剖结构、先天性免疫、细菌生物膜等有关,然而究竟是哪种因素是引起CRS的关键因素,有必要进行深入的研究。但目前研究显示,树突状细胞(dendritic cell,DC)、Th17/Treg失衡和micro RNAs(mi RNAs)是CRS发病的重要基础,然而其的发生机制尚未阐明,本研究将从DC-Th轴探讨mi RNA在CRS中的作用。DC是机体内一种专职抗原提呈细胞,因其表面有树突状突起而得名。DC在免疫系统中发挥极其重要的作用,不同的DC可以相互作用和调节。DC是如何影响免疫系统中其它细胞的功能呢?一方面,DC对外来微生物进行识别、摄取、加工处理和提呈,在天然免疫中发挥抗微生物感染的作用;另一方面,DC捕获抗原后能与初始T(na?ve T)细胞相互作用,激活na?ve T细胞,并通过分泌IL-17、IL-12、IL-10等细胞因子调控辅助性T(Th)细胞的诱导、分化,从而对启动和维系Th细胞反应起到决定性作用,调节获得性免疫应答。DC作为抗原递呈细胞,功能强大,能够阻止微环境中病原体的侵入,成熟的DC可以迁移到淋巴结而发生抗原递呈的作用,还可刺激T细胞增殖。在T细胞介导的机体细胞免疫中,CD4+Th细胞具有重要意义,na?ve T细胞在抗原提呈细胞的诱导下可分化为Th1细胞、Th2细胞、Th17细胞和T调节性细胞(T regulatory cells,Treg)发挥不同作用。Mi RNAs是一类单链的非编码RNA分子,长约19-22核苷酸,能够干预靶基因沉默的转录后调控和抑制目标m RNA翻译蛋白质。因此,mi RNAs是一类有望调节上气道炎症的微小分子,也就是在CRS中同样发挥作用。借助以上关于mi RNAs在各慢性炎症性疾病中作用以及mi RNA在DC中作用的研究结果,我们推测在CRS发病机制中DC-Th轴的异常必然有相当数量的mi RNA参与其中。然而,参与DC-Th轴异常的mi RNA表达谱具体如何?不同亚类的CRS是否存在共有和特异的差异表达mi RNA?mi RNA对DC-Th轴调控的机制如何?是否可以通过干预差异表达mi RNA调控DC-Th轴的平衡,最终达到疾病预防和治疗的目的?为了更有效的治疗CRS,有必要探索以上科学问题。本研究以CRS患者为研究对象,以DC-Th轴为主线,以mi RNA对DC的调控为切入点,明确CRS患者DC相关的mi RNA表达谱,探讨mi RNA对DC-Th轴的调控作用,以期从DC层面阐明CRS中Th细胞失衡的上游分子事件及调控机制,为CRS发病机制的研究及临床防治策略的建立提供新的靶点和新的思路。第一部分不同类型CRS患者外周血中DC表型分布的研究目的:采用流式细胞术(flow cytometry,FCM)检测DC在CRS外周血中的表型、数量、分布特征,比较它们之间的差异,观察DC在CRS发病机制中的作用。方法:收集2014年7月-2015年8月67例CRS患者,参照EPOS2012的诊断标准分为4个组:(1)正常对照组:选择单纯鼻中隔偏曲患者(13例);(2)CRSs NP:为不伴鼻息肉的CRS患者(18例);(3)atopic CRSw NP:伴变应性体质的慢性鼻-鼻窦炎伴鼻息肉患者(18例);(4)non-atopic CRSw NP:不伴变应性体质的慢性鼻-鼻窦炎伴鼻息肉患者(18例)。采用淋巴细胞分离液密度梯度离心法获得外周血单个核细胞(peripheral blood mononuclear cells,PBMC),再用免疫磁珠法分离得到CD+14单核细胞,在白细胞介素-4(interleukin-4,IL-4)和粒细胞集落刺激生物因子(granulocyte-macrophage colony stimulating Factor,GM-CSF)诱导培养后得到DCs,通过流式细胞术,检测CRS外周CD80、CD83、CD1a和CD86(DC成熟和活化标志)的表达。评价不同类型CRS外周血DC的表型、数量及分布特征。结果:CRS患者外周血中DC的分布情况:从FCM散点图可以看出成熟DCs的阳性比例:control 38.98%;CRSs NP 57.77%;atopic CRSw NP 84.87%;non-atopic CRSw NP 82.94%。同对照组相比,CRS中成熟DCs均高于对照组,而息肉组中成熟DCs高于非息肉组,伴有变应性体质的明显高于不伴有变应性体质。P0.05,差异有统计学意义。结论:DCs在CRS外周血中增多,DC在CRS患者的发病机制中可能扮演着重要的角色;而atopic组表现为更多的DC分布,提示变应性因素可能促使DC增多。因此,对于伴有变应性体质的CRS患者,除了手术治疗外,还应积极抗过敏治疗。第二部分确定CRS患者外周血DC的mi RNA表达谱目的:通过对CRS患者样本外周血中DC提取RNA后进行基因芯片检测,再用实时荧光定量PCR技术进一步验证芯片结果,确定出CRS患者外周血中DC的mi RNA表达谱,并进行定量分析,筛选差异表达mi RNA。评价CRS患者不同亚型CRS共有和特有的差异表达mi RNA。旨在探讨筛选出具有代表性的差异表达mi RNA在CRS发生发展中的作用。方法:分别收集3个组的CRS患者及对照组样本的外周血,先分离PBMC,予以免疫磁珠法分离CD14+的单核细胞,予以脂多糖(Lipopolysaccharide,LPS)诱导为成熟的DCs;再利用Trizol裂解蛋白之后,酚-氯仿反复抽提,提取DC中总RNA,采用mi RNA基因芯片分析技术进行检测,再用实时荧光定量PCR技术进一步验证芯片结果,确定出CRS患者外周血中DC的mi RNA表达谱,并进行定量分析,筛选差异表达mi RNA。结果:Mi RNA基因芯片结果显示:同对照组相比,不同类型的8CRS患者外周血中DCs有不同的差异表达的mi RNA,在这3种类型的CRS中共有31种差异表达的mi RNAs,其中有5种mi RNAs表达是上调,有25种mi RNAs表达是下调,而mi RNA-1290表达在CRSs NP中是下调,在atopic CRSw NP和non-atopic CRSw NP中是上调。结论:通过对3种类型的CRSmi RNA基因表达的比较,我们推测差异表达的mi RNA有可能通过调节DCs来干预CRS的发病机制,这些mi RNAs有可能是治疗CRS的一个新靶点。第三部分从DC-Th轴探讨mi R-150-5P在CRS中的作用目的:通过DC与原始T细胞(na?ve T细胞)共培养后,利用FCM检测T细胞的增殖情况和培养后上清液的IL-17的浓度情况,从DC-Th轴探讨mi R-150-5P在CRS中的表达,评价在CRS中mi R-150-5P引起Th17/Treg失衡的上游事件和它的免疫调节因素。方法:从不同类型CRS外周血中分离的DCs,诱导成熟后,转染mi R-150-5P模拟剂/抑制剂(mimic/inhibitor)后,与CFSE标记的同源异体的na?ve T细胞共培养5天(1:20),收集T细胞,予anti-CD3和anti-CD28刺激,通过FCM检测CFSE标记的T细胞比率,通过酶联免疫吸附试验(enzyme-linked immuno sorbent assay,ELISA)检测IL-17的浓度。结果:同未转染相比,转染mi R-150-5P mimic组的T细胞增殖和Il-17增加,而转染mi R-150-5P inhibitor组下降,有DCs共培养组高于未共培养组,CRS患者组高于正常对照组,P0.05,有统计学意义。结论:mi R-150-5P在CRS中的表达是上调表达,mi R-150-5P mimic促进T细胞增殖和IL-17分泌,mi R-150-5P inhibitor抑制T细胞增殖和IL-17分泌。mi R-150-5P通过DC-Th轴影响CRS发病的。第四部分预测并验证CRS患者DC中mi R-150-5P可能作用的靶基因目的:探讨差异表达mi R-150-5P可能作用的靶基因及靶蛋白,以期探讨通过干预差异表达mi R-150-5P对CRS疾病预防和治疗的意义。方法:采用生物信息学分析软件Target Scan、Pic Tar、Mi Randa和mi RBase Targets对与DC分化、成熟和功能相关的mi R-150-5P靶基因进行靶点预测,通过蛋白质免疫印迹试验(Western blot,WB)验证靶蛋白,通过荧光素酶报告试验,将靶蛋白3’UTR区域克隆至含虫荧光素酶报告基因3’UTR区,同时合成mi R-150-5P片段将二者共转染HEK293细胞(1x104),孵育48h后,对荧光素酶活性进行检测,验证该靶基因是否为mi R-150-5P的靶基因。结果:通过生物学软件预测早期生长反应蛋白2(early growth response 2,EGR2)mi R-150-5p的靶基因,并通过WB试验和荧光素酶报告试验验证EGR2是mi R-150-5P的靶基因。结论:Mi R-150-5p和它的靶基因EGR2在CRS的发病机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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R765

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