诱导型一氧化氮合酶mRNA在分泌性中耳炎中表达的意义

目的探讨分泌性中耳炎(secretory ototis media，SOM)患者中耳积液及外周血白细胞诱导型一氧化氮合酶(inducible nitric oxide synthase，iNOS)mRNA表达及在SOM发病过程中的意义，以及与细菌感染和免疫介导的关系。方法随机取45例SOM患者及30例健康人的外周血白细胞，并抽取SOM患者患耳中耳积液，用原位杂交方法检测iNOS—mRNA。结果健康人外周血白细胞未见iNOS—mRNA表达。SOM患者外周血白细胞iNOS—mRNA表达：急性组阳性率为63．64％，镜下阳性细胞率为60．3％；亚急性组阳性率为8．7％，镜下阳性细胞率为72．5％；SOM患者中耳积液iNOS—mRNA表达：急性组阳性率为45．45％，镜下阳性细胞率为80％；亚急性组阳性率为52．17％，镜下阳性细胞率为84％。SOM患者外周血白细胞及中耳积液中iNOS—mRNA表达高度增强，其中在急性组外周血白细胞中表达显著高于亚急性组，而中耳积液iNOS—mRNA表达阳性率及阳性细胞率在急性组与亚急性组中表达强度无显著性差异。结论诱导型一氧化氮合酶(iNOS)及诱导型一氧化氮合酶——氧化氮(iNOS—NO)通路在SOM的发病、中耳积液的形成过程中可能起着重要作用。     

变应性鼻炎动物模型血一氧化氮和鼻黏膜一氧化氮合酶的动态检测

目的探讨在变应性鼻炎（allergic rhinitis,AR）发病及应用抗过敏药物治疗过程的不同阶段,变应性鼻炎动物模型血一氧化氮（Nitric Oxide,NO）水平和鼻黏膜一氧化氮合酶（Nitric-Oxide Synthase,NOS）表达的动态变化。方法选用健康SD大鼠50只,将其随机分为5组：正常指标组、实验1组、实验2组、实验3组、治疗组。在不同阶段分别取血和鼻黏膜;检测血浆中NO含量及鼻黏膜匀浆液NOS含量。结果实验1组和实验3组动物的血浆NO及鼻黏膜匀浆上清液NOS含量均显著升高,实验2组的NO及NOS含量均无显著变化;治疗组NO含量降至正常指标组水平,NOS亦有部分下降。血浆NO与鼻黏膜NOS含量之间存在显著的正相关关系（r=0．770,P=0．013）。结论血浆NO及鼻黏膜NOS水平在AR造模的不同阶段有明显的波动性变化,常规抗过敏药物能有效抑制患病机体NO的产生。血NO含量的变化能较好地反应鼻黏膜NOS活性的改变。     

一氧化氮及其合酶在鼻息肉中的表达

目的：探讨一氧化氮(NO)及其合酶(NOS)在鼻息肉中的表达，以及NO在鼻息肉发病中的作用。方法：用免疫组织化学及原位杂交方法研究诱导型一氧化氮合酶(iNOS)及内皮型一氧化氮合酶(eNOS)在鼻息肉中的表达，同时用原位杂交方法研究iNOS mRNA的表达，并用硝酸还原酶法研究NO在鼻息肉中的产生情况。结果：鼻息肉组织中eNOS主要分布于上皮、腺体细胞及血管内皮细胞，其染色强度稍强于对照组。iNOS在上皮细胞呈现较强的阳性染色，在息肉组织内主要表达于散在的炎症细胞。结论：eNOS活性增高可能与鼻息肉发病中血管过度扩张、腺体病理性分泌增多等有关。iNOS生成的较高浓度的NO在鼻息肉的病理过程中可能起到促进炎症发展的作用。     

脾气虚型变应性鼻炎大鼠鼻黏膜IL-3mRNA的活性表达

目的 观察脾气虚型变应性鼻炎大鼠鼻黏膜组织白细胞介素-3(IL-3)mRNA的表达,探讨脾气虚与变应性鼻炎的病理相关性.方法 以卵清蛋白法和卵清蛋白加饮食不节法分别建立大鼠变应性鼻炎模型(AR组)和脾气虚变应性鼻炎模型(脾气虚AR组),分别取鼻黏膜组织,RT-PCR检测IL-3 mRNA表达水平,进行组间比较.结果 AR组鼻黏膜IL-3 mRNA表达水平11.85±0.82,显著高于健康对照组的6.85±0.49(P＜0.01);脾气虚AR组鼻黏膜IL-3 mRNA表达水平14.21±2.10,显著高于AR组(P＜0.01).结论 变应性鼻炎大鼠鼻黏膜组织IL-3 mRNA的表达增加, 脾气虚型变应性鼻炎大鼠者IL-3 mRNA表达活性进一步升高,IL-3活性表达水平可能是脾气虚型变应性鼻炎病情加重的一个重要因素.     

一氧化氮与变应性鼻炎

一氧化氮（nitric oxide,NO）是一种结构极为简单的无机小分子,也是一种极不稳定的生物自由基,是由L-精氨酸在一氧化氮合酶（nitric oxide synthase,NOS）催化下合成的生物活性物质,具有广泛的生物学作用。近年来研究发现鼻炎患者黏膜上皮、腺体及血管也均见NOS表达,其中腺体表达明显增高,提示NO在鼻黏膜腺体功能上起重要作用,并且发现变应性鼻炎影响着NO的水平变化。本文就近几年国内外研究者对NO和变应性鼻炎之间关系及可能的作用机制做一综述。     

富氢生理盐水对变应性鼻炎豚鼠诱导型一氧化氮合酶及其代谢产物水平的影响

目的　探讨富氢生理盐水（hydrogen-rich saline,HRS）对变应性鼻炎（AR）豚鼠iNOS及其代谢产物水平的影响及相关抗炎机制。方法　24只健康雄性豚鼠,随机分3组,对照组、AR组、HRS干预组。以卵清蛋白（OVA）致敏法建立AR豚鼠模型,HRS干预组致敏后HRS干预2周,对照组均同期使用等量生理盐水替代。观察各组豚鼠症状、外周血嗜酸性粒细胞（Eos）计数;分别检测血浆一氧化氮（nitric oxide,NO）水平与血清诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）表达水平;RT-PCR及Western blot检测鼻黏膜iNOS mRNA与蛋白的表达。结果　AR组豚鼠行为学评分及Eos计数较对照组高,HRS干预后比AR组行为学比分明显降低,差异有统计学意义。AR组血浆中NO含量较对照组高[（61.87±4.25）μmol/L vs（29.49±2.79）μmol/L],HRS干预组较AR组 低[（39.44±2.95）μmol/L vs（61.87±4.25）μmol /L]（P均<0.05）。AR组血清中iNOS含量较对照组明显增加[（292.13±10.95）IU/ml vs（19.72±2.04）IU/ml],HRS干预组较AR组明显下降,差异有统计学意义[（48.24±4.67） IU/ml vs（292.13±10.95）IU/ml]（P 均<0.05）。AR组豚鼠鼻黏膜中iNOS mRNA表达量较对照组高（0.76±0.05 vs 0.11±0.03）,HRS干预组较AR组低（0.21±0.04 vs 0.76±0.05）,差异均有统计学意义（P 均<0.05）。AR组豚鼠iNOS蛋白表达水平高于对照组,HRS干预后,iNOS蛋白量下降。结论　HRS减轻AR炎症,下调AR豚鼠iNOS-NO表达。     

变应性鼻炎大鼠鼻黏膜Bcl-2 mRNA及其蛋白的表达

目的 :探讨变应性鼻炎 (AR)大鼠鼻黏膜凋亡相关基因Bcl 2mRNA及其蛋白的表达 ,以进一步了解变应性鼻炎的发病机制。方法 :将 2 0只大鼠随机分为实验组和对照组 ,每组 10只。实验组以卵清蛋白腹腔注射致敏 ,继之鼻局部激发建立变应性鼻炎动物模型。取实验组与对照组动物鼻呼吸区黏膜 ,行Bcl 2mRNA原位杂交染色、Bcl 2与Bax蛋白免疫组化染色 ,并行苏木精 伊红染色以资对比。结果 :实验组Bcl 2mRNA与Bcl 2蛋白主要表达于鼻黏膜腺体细胞 ,其次是上皮层 ,鼻分泌物中可见大量呈强阳性反应的嗜酸性粒细胞 ,Bcl 2mRNA与Bcl 2蛋白表达水平显著高于对照组 (P <0 .0 5 ) ,Bax蛋白与Bcl 2蛋白表达部位一致 ,两组表达水平差异无统计学意义 (P >0 .0 5 )。结论 :变应性鼻炎大鼠鼻黏膜凋亡调控基因Bcl 2mRNA及其蛋白表达上调 ,是变应性鼻炎鼻分泌物增多及鼻黏膜上皮破坏的机制之一     

SOCS3和Eotaxin蛋白及其mRNA在变应性鼻炎鼻黏膜组织中的表达

目的观察细胞因子信号转导抑制因子3（suppressor of cytokime singnaling-3,SOCS3）、嗜酸性粒细胞趋化因子（Eotaxin）在变应性鼻炎鼻黏膜组织中的表达。方法采用RT—PCR检测24例变应性鼻炎（allergic rhinitis,AR）患者鼻黏膜组织（AR组）和22例单纯鼻中隔偏曲患者黏膜组织（对照组）中SOCS3 mRNA、Eotaxin mRNA的表达水平,采用免疫组织化学方法检测两组患者鼻黏膜组织中SOCS3、Eotaxin蛋白的表达情况,分析SOCS3、Eotaxin蛋白及mRNA表达的相关性。结果AR组鼻黏膜组织中SOCS3、Eotaxin蛋白及mRNA表达水平明显高于对照组（P〈0．01）,且AR鼻黏膜组织中SOCS3蛋白及mRNA的表达水平与Eotaxin蛋白及mRNA的表达呈正相关。结论SOCS3与Eotaxin蛋白及其mRNA的表达可能在变应性鼻炎的发生过程中起到了一定的作用。     

变应性鼻炎患者血清氧化应激状态研究

目的 检测变应性鼻炎(allergic rhinitis,AR)患者外周血血清中氧化应激标志物,探讨氧化应激反应在AR发病机制中的作用.方法 留取23例健康人、48例AR患者外周血血清,分别检测氧化应激标志物一氧化氮(nitric oxide,NO)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、总一氧化氮合酶(total nitric oxide synthase,TNOS),脂质过氧化产物丙二醛(malondidehyde,MDA),以及体内主要抗氧化酶超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)水平.采用SPSS 13.0软件对数据进行分析.结果 AR患者血清NO水平((x)±s,以下同)为[(97.92±73.42)μmol/L]较健康对照组[(64.04±29.54)μmol/L]明显升高(t=-0.281,P<0.05);iNOS与TNOS比值(0.51±0.11)较健康对照组(0.45±0.15)明显升高(t=-2.061,P<0.05);血清GSH-Px活性[(258.24±45.25)U/(ml·min)]较健康对照组[(215.11±47.62)U/(ml·min)]明显升高(t=-2.2349,P<0.05)).而AR患者血清SOD活性、MDA含量较健康对照组分别有升高和降低趋势,但差异无统计学意义(Z=-1.656,t=1.922,P值均>0.05).结论 氧化应激反应参与AR病理生理过程,而iNOS-NO通路在AR的发病过程中可能发挥着更为重要的作用.     

慢性鼻窦炎患者鼻一氧化氮/一氧化氮合酶表达水平与黏膜损伤水平的相关性研究

目的　探讨慢性鼻黏膜炎症中黏膜损伤程度与一氧化氮/一氧化氮合酶NO/NOS表达之间的关系。方法　选取连续进行鼻内镜手术的42例患者及5名正常人,进行常规的术前评估及鼻NO的测量;并在内镜手术中进行黏膜组织取材,固定后进行HE染色及NOS的免疫组化染色;评估内皮型NOS（eNOS）及诱导型NOS（iNOS）的表达程度、黏膜损伤的程度。结果　iNOS和eNOS在钩突（筛泡、中鼻甲及息肉组织）的黏膜层、黏膜下层及腺体中的表达分级和鼻NO浓度无明显相关性（P 均>0.05）;且iNOS与eNOS在黏膜各层的总表达情况与鼻NO浓度亦无明显相关性（P >0.05）。鼻黏膜中炎症细胞的浸润程度和鼻NO浓度无明显相关（P >0.05）。而鼻黏膜慢性炎症损伤程度和鼻NO表现出明显的负相关关系。结论　鼻NO浓度和鼻黏膜炎症损伤指标之间具有明显相关关系。因此,鼻黏膜炎症中鼻黏膜的炎症状态可能是调节鼻NO浓度的主要因素;鼻黏膜上皮可能是影响鼻NO的主要部位。     

变应性鼻炎患者外周血白细胞及鼻黏膜诱导型一氧化氮合酶mRNA表达的意义

OBJECTIVE: To investigate the relationship between expression of leukocyte inducible nitric oxide synthase (iNOS)-mRNA and allergic rhinitis (AR). METHOD: Thirty-five patients with AR and 30 healthy controls were included in this study. Expression of iNOS-mRNA in peripheral blood leukocyte was detected by in situ hybridization. NO in plasm was measured by nitrate reductase. Expression of iNOS-mRNA in nasal mucosal was detected in 8 patients with AR and 6 healthy controls. RESULT: No expression of leukocyte iNOS-mRNA in healthy controls was found. In AR patients, the positive cells were significantly increased, the positive rate reached 40.82%. Expression of iNOS-mRNA was localized at the epithelium, gland and macrophage in healthy controls. Hyperplasia and expression of iNOS-mRNA increased at epithelium, gland and macrophage in the AR patients(t = 23.17, P < 0.001). The level of plasm NO in AR group was higher than that in healthy control group (t = 27.89, P < 0.001). CONCLUSION: There is a relationship between expression of leukocyte iNOS-mRNA and the level of plasm NO in AR patients. The study provides an easy method of in situ hybridization for detecting some signal in body.     

Toll样受体-4介导诱发型一氧化氮合酶mRNA在鼻黏膜上皮细胞中的表达

目的 :探讨Toll样受体 4 (TLR 4 )对诱发型一氧化氮合酶 (iNOS )在鼻黏膜上皮细胞中表达的上调作用。方法 :应用核酸分子原位杂交技术检测 30例慢性鼻窦炎患者 (鼻窦炎组 )和 2 1例健康者 (对照组 )鼻黏膜上皮细胞中TLR 4和iNOSmRNA的表达。结果 :TLR 4和iNOSmRNA在鼻窦炎组鼻黏膜上皮细胞中的表达 ,分别为 (0 .14 33± 0 .0 184 0 ) /A和 (0 .136 8± 0 .0 0 76 5 ) /A ,分别与对照组 [(0 .10 2 4± 0 .0 1133) /A和 (0 .0 72 4± 0 .0 136 4 ) /A ]比较 ,差异有统计学意义 (均P <0 .0 1) ,且二者表达呈正相关 (r =0 .4 35 ,P <0 .0 1)。结论 :鼻黏膜上皮细胞通过TLR 4识别病原微生物 ,上调iNOSmRNA的表达 ,增加NO的合成 ,以杀伤、清除病原菌 ,增强宿主防御和免疫应答。     

缺氧对鼻粘膜上皮细胞中诱发型一氧化氮合酶的影响及其意义

目的 :探讨鼻粘膜上皮细胞应答局部组织缺氧 ,合成分泌诱发型一氧化氮合酶 (iNOS)的变化 ,以及地塞米松对此的影响及意义。方法 :将人鼻粘膜上皮细胞在常氧和缺氧状态下进行无血清原代细胞培养 ,并加入不同浓度的地塞米松共同孵育 ,采用流式细胞仪观察常氧和缺氧条件下上皮细胞动力周期的变化 ,采用原位杂交的方法检测iNOSmRNA的变化。结果 :缺氧条件下上皮细胞的动力周期延长 ;在缺氧 3h后iNOSmRNA水平开始升高 ,12h达高峰 ,2 4h后下降 ,4 8h几乎消失 ;加入地塞米松后 ,降低这种升高的水平。但 4 μg L以上的浓度 ,并不进一步降低被缺氧升高的iNOSmRNA水平。结论 :缺氧诱发鼻粘膜上皮细胞合成高水平的iNOSmR NA ,应用一定浓度的地塞米松能降低这种作用     

Reduction of neuronal and inducible nitric oxide synthase gene expression in patients with cystic fibrosis

As a consequence of diminished nitric oxide synthase (NOS) protein concentration, the airway concentration of nitric oxide (NO) is reduced in patients with cystic fibrosis (CF). This appears to lead to a reduced elimination of such microorganisms as Pseudomonas aeruginosa. The objective of this study was to analyze whether inducible (iNOS), endothelial (eNOS) and neuronal (bNOS) NOS are reduced at mRNA level and if so whether this is caused directly by the defective CF transmembrane conductance regulator (CFTR). Nasal polyps from three patients with CF and four otherwise healthy patients were obtained. The expression of the three NOS isoenzymes was quantified using real-time PCR. The iNOS expression was assessed in colon carcinoma cells (CaCo) transfected with a normal and a mutated (DeltaF508) CFTR. In CF patients, iNOS mRNA expression was 10- to 20-fold and bNOS gene expression was one-fifth to one-tenth that in control patients (P < 0.001). In CaCo cells, iNOS gene expression under basal and endotoxin-stimulated conditions did not differ between cells transfected with a mutated CFTR and those transfected with an intact CFTR. This observation suggests that cystic fibrosis is associated with reduced iNOS and bNOS gene expression in nasopharyngeal tissue, possibly disturbing the barrier against infective agents already at the site of entrance. Received: 7 March 2001 / Accepted: 26 September 2001     

Increased exhaled nitric oxide and its oxidation metabolism in eosinophilic chronic rhinosinusitis

Objective

Monitoring of fractional concentrations of exhaled nitric oxide (FeNO) has become a reliable marker of inflammation in human nose and paranasal sinuses. However, it is still unknown to what extent nasal NO levels contribute to the pathology of chronic rhinosinusitis (CRS). In the present study, we aimed to examine FeNO levels and the underlying mechanism of NO production and metabolism in patients with eosinophilic chronic rhinosinusitis (ECRS) and non-ECRS.

Methods

Thirty-three untreated ECRS patients, 16 non-ECRS patients, and 38 normal subjects were enrolled in this cross-sectional study of FeNO levels. Oral and nasal FeNO levels were measured before treatment using an electrochemical NO analyzer (NObreath^®) with a nose adaptor. The mRNA expression of three nitric oxide synthase (NOS) isoforms, interleukin-5 (IL-5), and transforming growth factor-beta (TGF-β) in the ethmoid sinus mucosa and nasal polyps were analyzed by real-time PCR. Immunohistological localization of inducible NOS (iNOS) and nitrotyrosine (NT), a marker for oxidized NO metabolites, was also examined.

Results

ECRS patients showed significantly higher oral FeNO levels compared to non-ECRS patients and normal subjects (mean values, 47.6, 13.5, and 15.3 ppb, respectively). Nasal FeNO levels of the non-ECRS patients (30.5 ppb) were significantly lower than those of the ECRS patients (53.9 ppb) and normal subjects (45.5 ppb). Positive correlations existed between the blood eosinophil percentage and FeNO levels in ECRS patients. Histologically, ECRS patients showed higher eosinophil accumulation in the ethmoid mucosa than non-ECRS patients (103.1 vs. 16.3 cells/HPF). Real-time PCR analysis showed significant upregulation of iNOS and IL-5 mRNA expression in the ethmoid mucosa of the ECRS patients compared to those of non-ECRS patients. Positive iNOS immunoreactivity was observed in ciliated epithelial cells, submucosal glands and associated inflammatory cells in both groups. NT immunoreactivity was detected in the epithelium and around inflammatory cells. Intense NT staining was co-localized with eosinophil accumulation and ECRS patients showed significantly higher rates of NT-positive cells than non-ECRS patients.

Conclusion

A combination of oral and nasal FeNO measurement is a valid marker for the classification and definition of different CRS subtypes in Japan. Higher levels of oral and nasal FeNO in ECRS patients may reflect the persistence of eosinophilic inflammation in sinus mucosa with concomitant iNOS upregulation and accompanying deposition of oxidized NO metabolites.     

变应性鼻炎患者一氧化氮检测与分析

目的 检测变应性鼻炎患者鼻分泌物中一氧化氮(NO)的含量,并分别与慢性鼻炎患者和正常对照者鼻分泌物中NO水平进行比较。方法 测定了50例变应性鼻炎患者和40例慢性鼻炎患者及40例正常对照组鼻分泌物NO水平。结果 变应性鼻炎患者体内NO合成较正常对照组及慢性鼻炎患者高。结论 NO参与了变应性鼻炎的发病过程,对其机理的研究有利于变应性鼻炎的治疗。