骨膜蛋白在慢性鼻—鼻窦炎不同表型中差异表达的实验研究

发布时间：2018-01-19 01:18

本文关键词： 慢性鼻-鼻窦炎骨膜蛋白炎症重塑　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景和目的:依据EPOS2012,鼻-鼻窦炎被定义为:鼻腔和鼻窦的炎症,具有两个或两个以上的症状特征,其中一个必备条件是鼻塞/鼻堵,或鼻漏(前后鼻孔滴漏),可以伴或不伴面部疼痛/压迫感、嗅觉减退或丧失;并且鼻内镜检查所见:鼻腔息肉,并且/或伴有主要来源于中鼻道的黏脓性分泌物,并且/或伴有主要位于中鼻道的黏膜水肿或阻塞;或CT检查:伴有窦口鼻道复合体和/或鼻窦的黏膜改变1。慢性鼻-鼻窦炎的症状持续时间不少于12周,并且症状未彻底消失。在临床上,可将慢性鼻-鼻窦炎(CRS)分为慢性鼻-鼻窦炎伴鼻息肉(CRSwNP)和慢性鼻-鼻窦炎不伴鼻息肉(CRSsNP),对于慢性鼻-鼻窦炎伴鼻息肉,有学者依据鼻息肉中浸润细胞的种类和数量,把慢性鼻-鼻窦炎伴鼻息肉分为嗜酸性粒细胞型、中性粒细胞型、非嗜酸性粒细胞非中性粒细胞型2。不同的慢性鼻-鼻窦炎表型,其各自的内在病理生理学特点存在差异,这种差异可能预示不同的发病机制,识别不同的发病机制可通过特定的生物标志物来完成,已经发现某些生物学标志物,如血液中特异性IgE、金黄色葡萄球菌内毒素IgE等,可作为慢性鼻-鼻窦炎的不同内在型的生物标志物3,并且针对不同内在型的标志物可以选择个体化的治疗4,5。研究报道骨膜蛋白(Periostin)是嗜酸性粒细胞性哮喘患者呼吸道炎症的全身性生物标志物6,7,参与哮喘患者呼吸道黏膜上皮下组织纤维化等结构重塑8,9。上下呼吸道常常同时存在炎症10,组织病理学研究发现与哮喘患者下呼吸道结构重塑相一致,在慢性鼻-鼻窦炎患者鼻腔鼻窦组织中也存在结构重塑11。由此我们推测,在慢性鼻-鼻窦炎的组织中,可能存在骨膜蛋白的表达,这种表达在不同的慢性鼻-鼻窦炎表型可能具有差异,这种差异对于认识慢性鼻-鼻窦炎的内在分型具有重要意义,与慢性鼻-鼻窦炎的发病机制有一定的关系。在哮喘患者中,血清中升高的骨膜蛋白与一种特殊的哮喘表型-嗜酸性粒细胞型哮喘有一定关系,并且经常合并有阻塞性肺功能障碍和鼻部疾病12,在Th2型(IL-5、IL-13高表达)、伴有较高的嗜酸性粒细胞的哮喘表型中,与血中嗜酸性粒细胞数、痰中嗜酸性粒细胞相比,血清中骨膜蛋白对于检测气流受限具有更高的敏感性13,血清中骨膜蛋白可以作为哮喘的生物标志物,主要表现在两个方面:一是作为Ⅱ型免疫反应的生物标志物代替指标,二是作为反映14组织重塑或纤维化的生物标志物14。已有研究发现在慢性鼻-鼻窦炎伴鼻息肉中,59. 6%存在嗜酸性粒细胞(EOS)浸润,嗜酸性粒细胞浸润在慢性鼻-鼻窦炎(CRS)15的病理生理过程中处于中心位置15。在嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉中,骨膜蛋白的表达可能与嗜酸性粒细胞的浸润有一定的关系,进而可能与嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉的形成有关。目的:我们的研究旨在观察骨膜蛋白在慢性鼻-鼻窦炎不同表型的表达,分析其在慢性鼻-鼻窦炎病理生理过程中的作用,为慢性鼻-鼻窦炎的内在分型提供可能的依据。方法:选择住院患者,分为慢性鼻-鼻窦炎不伴鼻息肉组(CRSsNP、19例)、慢性鼻-鼻窦炎伴鼻息肉组(CRSwNP、36例)、鼻中隔偏曲组(DNS、15例)。分别留取慢性鼻-鼻窦炎不伴鼻息肉组的筛窦黏膜和慢性鼻-鼻窦炎伴鼻息肉组患者的筛窦黏膜组织与鼻息肉组织、鼻中隔偏曲患者的下鼻甲组织作为标本,对慢性鼻-鼻窦炎伴鼻息肉组标本进行HE染色,依据嗜酸性粒细胞的浸润情况,将慢性鼻-鼻窦炎伴鼻息肉组分为嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组(ECRSwNP)和非嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组(NonECRSwNP),对四组标本进行骨膜蛋白免疫组织化学染色(IHC)、实时定量逆转录-聚合酶链式反应(RT-PCR),观察骨膜蛋白分子及mRNA在四组标本的表达;通过酶联免疫吸附试验(ELISA)法检测四组患者血清中骨膜蛋白的浓度。并对局部组织中免疫组织化学染色评分、骨膜蛋白mRNA、嗜酸性粒细胞浸润和血清中骨膜蛋白浓度、血液中嗜酸性粒细胞比例分别进行各指标间相关性分析。结果:骨膜蛋白免疫组化染色显示,骨膜蛋白在四组标本中的表达主要位于鼻黏膜上皮的基底膜、上皮下的纤维组织。慢性鼻-鼻窦炎不伴鼻息肉组(筛窦黏膜)、嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组(筛窦黏膜、鼻息肉)、非嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组(筛窦黏膜、鼻息肉)和鼻中隔偏曲组(下鼻甲黏膜)的免疫组化染色评分分别为3.19±0.98、3. 90±0. 42/4.17士0.52、3.20±0.86/3.32±0.84和2.44±0.86,慢性鼻-鼻窦炎不伴鼻息肉组、嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组以及非嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组评分高于鼻中隔偏曲组,差异具有统计学意义(P0. 05、P0.01、P0. 01)。嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组高于其他三组,差异具有统计学意义(P0. 01)。慢性鼻-鼻窦炎不伴鼻息肉组、非嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组之间相比较,差异无统计学意义(P0. 05)。通过酶联免疫吸附方法测得四组患者血清中的骨膜蛋白浓度,慢性鼻-鼻窦炎不伴鼻息肉组、嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组、非嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组和鼻中隔偏曲组血清中骨膜蛋白浓度分别为47962. 77±14307. 30、72138. 91 ± 21722.43、43012.45 ± 10314. 95 和 31337.81 士8181. 02pg/ml。嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组明显高于其他三组,差异具有统计学意义(P0. 01),鼻中隔偏曲组低于其他三组,差异具有统计学意义(P0. 01),慢性鼻-鼻窦炎不伴鼻息肉组与非嗜酸性慢性鼻-鼻窦炎伴鼻息肉组相比无明显差异(P0. 05)。四组患者鼻黏膜组织中均有骨膜蛋白mRNA,慢性鼻-鼻窦炎不伴鼻息肉组、嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组、非嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组和鼻中隔偏曲组鼻黏膜组织骨膜蛋白mRNA分别为 0.351156 ±0.250896、0.477044 ±0.505552、0.326074 ±0.412194和0.340352±0. 304540,与鼻中隔偏曲组相比,慢性鼻-鼻窦炎不伴鼻息肉组、嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组、非嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组鼻黏膜组织中骨膜蛋白mRNA升高,但差异无统计学意义(P0.05)。在嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉组,鼻息肉组织中骨膜蛋白分子的表达与局部组织(筛窦黏膜、鼻息肉)中嗜酸性粒细胞的浸润具有一定相关性(P0. 01,P0. 05),血清骨膜蛋白浓度与血中嗜酸性粒细胞比例具有一定的相关性(P0. 01)。在四组标本中,局部组织中骨膜蛋白分子的表达与血中嗜酸性粒细胞比例无明显相关性(P0. 05)。筛窦黏膜骨膜蛋白mRNA的表达与局部组织中嗜酸性粒细胞浸润、血中嗜酸性粒细胞比例无明显相关性(P0. 05)。结论:1、骨膜蛋白作为炎性介质之一,参与慢性鼻-鼻窦炎的慢性炎症过程。2、骨膜蛋白参与嗜酸性粒细胞性慢性鼻-鼻窦炎伴鼻息肉的发病过程,这与嗜酸性粒细胞的浸润具有一定的正相关;3、骨膜蛋白参与慢性鼻-鼻窦炎不同分型的组织重塑过程。
[Abstract]:Background and objective: Based on the EPOS2012, rhinosinusitis is defined as inflammation of the nasal cavity and paranasal sinus, with two or more than two of the symptoms, which is a necessary condition for nasal / nasal obstruction, rhinorrhea (or nasal drip), with or without facial pain / oppression, loss or loss of smell; and endoscopic findings: nasal polyps, and / or with mainly from the nose of the sticky purulent secretions, and / or with mainly located in the middle meatus mucosa edema or obstruction; or CT examination: with the ostiomeatal complex and / or sinus mucosa of 1. chronic rhinitis sinusitis symptoms duration of not less than 12 weeks, and the symptoms did not disappear. In clinic, the chronic rhinosinusitis (CRS) for chronic sinusitis with nasal polyps (CRSwNP) and chronic nasal sinusitis without nasal polyps (CRSsNP), for chronic rhinosinusitis with the nasal According to different types of polyps, some scholars and the number of cell infiltration in nasal polyps, the chronic sinusitis with nasal polyps were divided into eosinophils, neutrophils, non eosinophilic non neutrophil type 2. different phenotypes of chronic rhinosinusitis, their intrinsic pathophysiological characteristics the difference, this difference may indicate different etiology, pathogenesis can identify different specific biomarkers to complete, have found some biological markers, such as specific IgE in the blood, Staphylococcus aureus endotoxin IgE, can be used as different internal chronic rhinosinusitis biomarkers of 3. And you can select the individualized treatment of 4,5. research reports for internal Periostin symbol of different types of animals (Periostin) is eosinophilic airway inflammation in patients with asthma and systemic biomarkers 6,7, In patients with respiratory tract mucosa on asthma subcutaneous tissue fibrosis and other structural remodeling of 8,9. lower respiratory tract inflammation in 10 often exist at the same time, research findings were consistent with the respiratory tract of patients with asthma under tissue remodeling pathology in patients with chronic rhinosinusitis nasal tissues also exist in the remodeling of 11. we conclude that in patients with chronic rhinosinusitis group in the expression of Periostin may exist, this expression in chronic nasal sinusitis may have different phenotypic differences, this difference has important significance for understanding the intrinsic type of chronic nose - sinusitis, have a certain relationship with the pathogenesis of chronic rhinosinusitis. In patients with asthma and elevated serum Periostin with a special asthma phenotype - eosinophilic asthma have a certain relationship, and often associated with obstructive pulmonary dysfunction and nasal disease in 12, Th2 Type (IL-5, high expression of IL-13), with a higher eosinophilic asthma phenotype, the number of eosinophils in blood and sputum eosinophils, compared with periosteum proteins in serum for detection of airflow limitation has a higher sensitivity of 13, periosteal proteins in serum can be used as biomarkers for asthma that is mainly manifested in two aspects: one is the type of immune response biomarkers instead of two indicators, as reflected in 14 tissue remodeling or fibrosis biomarkers of 14. have been found in chronic sinusitis with nasal polyps, 59.6% eosinophilic infiltration of eosinophil infiltration (EOS), acid particle cells in patients with chronic rhinosinusitis (CRS) 15 pathophysiological processes in the center position of 15. in eosinophilic chronic rhinosinusitis with the nasal polyps, the expression of Periostin may be and eosinophil infiltration The relationship between, and may be related to the formation of eosinophilic chronic rhinosinusitis with the nasal polyp. Objective: our study aimed to investigate the expression of Periostin in chronic rhinosinusitis with different phenotypes, in the analysis of the pathophysiology of chronic rhinosinusitis, may provide a basis for the internal classification of chronic rhinitis sinusitis. Methods: inpatients were divided into chronic sinusitis without nasal polyps (19 CRSsNP cases), chronic sinusitis with nasal polyps (group CRSwNP, 36 cases), nasal septum group (DNS, n = 15). The organization of ethmoid mucosa tissue and nasal polyps were collected chronic sinusitis without nasal polyps group and ethmoid sinus mucosa of chronic sinusitis with nasal polyps patients, patients with nasal septum deviation in inferior turbinate tissues as specimens of chronic rhinosinusitis with the nasal polyp group specimens were stained with HE, on the basis of eosinophil The infiltration, the chronic sinusitis with nasal polyps were divided into eosinophilic chronic rhinosinusitis with the nasal polyp group (ECRSwNP) and non eosinophilic chronic rhinosinusitis with the nasal polyp group (NonECRSwNP), immune tissue of the four groups were Periostin staining (IHC) and real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), to observe the expression of Periostin protein molecules and mRNA in the four groups; by enzyme-linked immunosorbent assay (ELISA) method to detect the concentration of serum protein was four. And the local tissue free immunohistochemical staining scores, Periostin Periostin mRNA, eosinophil the concentration of eosinophil infiltration and the serum, blood eosinophil percentage were analyzed the correlation between indexes. Results: Periostin immunohistochemical staining showed that the expression of Periostin in four groups were mainly located in the nose The basement membrane of the epithelium, subepithelial fibrous tissue. Chronic sinusitis without nasal polyps (ethmoid mucosa), eosinophilic chronic rhinosinusitis with the nasal polyp group (ethmoid mucosa, nasal polyps), non eosinophilic chronic rhinosinusitis with the nasal polyp group (ethmoid mucosa), nasal polyps and nasal septum deviation group (inferior turbinate) immunohistochemical staining scores were 3.19 + 90 0.98,3. + 0. 42/4.17 + 0.52,3.20 + 0.86/3.32 + 0.84 and 2.44 + 0.86, chronic sinusitis without nasal polyps, eosinophilic chronic rhinosinusitis with the nasal polyp group and non eosinophilic chronic rhinosinusitis with the nasal polyp group was higher than that of nasal septum group, the difference was statistically significant (P0. 05, P0.01 01, P0.). Eosinophilic chronic rhinosinusitis with the nasal polyp group than the other three groups, the difference was statistically significant (P 0.01). Chronic sinusitis without nasal polyps, non eosinophilic chronic sinusitis with nasal polyps between group comparison, no statistically significant difference (P0.. 05) by ELISA method to measure the concentration of Periostin four groups in the serum of patients with chronic rhinosinusitis without. Nasal polyps, eosinophilic chronic rhinosinusitis with the nasal polyp group, non eosinophilic chronic sinusitis with nasal polyps and nasal septum deviation group Periostin protein concentrations were 47962.77 + 14307. and 3072138.91 + 21722.4343012.45 + 10314.95 and 31337.81 + 8181. 02pg/ml. eosinophilic chronic rhinosinusitis with nasal polyps group was significantly higher than that of the other three groups in the serum, the difference was statistically significant (P0. 01), nasal septum group was lower than that of the other three groups, the difference was statistically significant (P0. 01), chronic nose sinusitis without The nasal polyp group and non eosinophilic chronic rhinosinusitis with the nasal polyp group showed no significant difference (P0. 05). Four groups of patients had periosteal protein mRNA in nasal mucosa, chronic nasal sinusitis without nasal polyps, eosinophilic chronic rhinosinusitis with the nasal polyp group, non eosinophilic cell chronic rhinosinusitis with periosteal protein mRNA in nasal polyps and nasal septum nasal mucosa tissue were 0.351156 + 0.250896,0.477044 + 0.505552,0.326074 + 0.412194 and 0.340352 + 0.304540, compared with the deviation of nasal septum, chronic nasal sinusitis without nasal polypus group, eosinophilic chronic rhinosinusitis with nasal polyps group, non eosinophilic chronic rhinosinusitis with periosteal protein mRNA in nasal polyps in the nasal mucosa increased, but the difference was not statistically significant (P0.05). In eosinophilic chronic rhinosinusitis with breath Meat group, expression of Periostin protein molecules in nasal polyps and local tissue (ethmoid mucosa, nasal polyps) is a relationship between the infiltration of eosinophils in the eosinophil (P0. 01, P0. 05), serum Periostin concentration and blood eosinophil proportion has certain correlation (P0. 01) in the four groups. The expression of blood specimens, and periosteal protein molecules in the local tissue eosinophil ratio had no significant correlation (P0. 05). The expression of mRNA protein and the periosteum of ethmoid mucosa tissue eosinophil infiltration, blood eosinophil percentage had no significant correlation (P0. 05). Conclusion: 1, periosteal protein is one of the inflammatory mediators,.2 chronic inflammatory process in chronic rhinosinusitis, Periostin protein involved in the pathogenesis of eosinophilic chronic rhinosinusitis with the nasal polyps, and the infiltration of eosinophils has certain positive correlation; 3, Periosteum protein participates in the process of tissue remodeling in different types of chronic rhinosinusitis.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R765

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