【作者】 安云芳；

【导师】 赵长青；

【作者基本信息】 山西医科大学 ， 耳鼻咽喉学， 2009， 博士

【摘要】 本课题选肥大细胞基因缺陷鼠和同品系正常鼠作为研究对象,在变态反应性鼻炎模型建立细菌感染性鼻窦炎模型。借助三维测量法定量分析鼻窦粘膜的炎性细胞浸润、肥大细胞脱颗粒、致病菌与鼻窦粘膜的相互构筑关系等,同时进行鼻窦粘膜上皮线粒体、线粒体内自嗜体及上皮与致病菌的关系等超微结构分析。通过测定鼻窦粘膜表面短路电流、以及分析“辣根过氧化物酶”跨细胞转运的速率等,评估鼻窦粘膜的上皮屏障功能。借助微渗泵进行化学性干预。本研究旨在阐明:呼吸道变态反应性炎症的发病并非首先是Th1/Th2及相关的细胞因子失平衡所致,而可能首先是肥大细胞基因表达异常所致;由肥大细胞介导的鼻变态反应性炎症是诱发鼻窦炎的关键因素。研究分为四部分,主要结果和内容如下:一、肥大细胞基因缺陷鼠急性细菌性鼻窦炎模型的构建目的:探讨肥大细胞缺失c-kit基因突变纯合子鼠(简称WADm/m小鼠)和和其同源的c-kit基因突变杂合子鼠(简称WADm/+小鼠)细菌性鼻窦炎与变应性鼻炎的复合模型的感染过程及二者之间的差别。材料与方法健康清洁级Wadm/m小鼠(A、C、E、G)和Wadm/+小鼠(B、D、F、H)各40只,每组随机分为4组:A组、B组(卵清蛋白+生理盐水);C组、D组(卵清蛋白+肺炎链球菌);E组F组(PBS+肺炎链球菌);G组、H组(PBS+生理盐水),每组各10只。①实验第0～8天A、B、C和D组用10%卵清蛋白(ovalbumin)腹腔注射、第9～16天6%卵清蛋白鼻腔局部激发制成AR模型;其余四组用PBS替代。②在AR基础上于实验第12天C、D、E、F组鼻腔滴入肺炎链球菌ATCC 49619(1.2×109CFU/ml);A、B组和G、H组用生理盐水代替。细菌滴鼻后分别于接种2d (各2只)、5d (各4只)、l0d (各2只)、14d (各2只)处死动物,对照组于第5天处死.,处死前各组动物内眦静脉采血,收集鼻腔灌洗液,以间接酶联免疫吸附试验(ELISA)检测血清白细胞介素-5(IL-5,IL-13)水平;取鼻面骨,石蜡包埋,连续切片,HE染色和甲苯胺蓝染色,计算机辅助光学显微镜观察肥大细胞浸润情况,计算每平方毫米鼻窦黏膜中多形核中性粒细胞(,PMN)数目。处死前作鼻腔灌洗培养,头颅石蜡包埋,连续切片,HE染色,计算机辅助光镜观察,计算窦腔内中性粒细胞集落所占窦腔的百分率和每平方毫米窦腔粘膜中浸润的多形核白细胞数。结果:WADm/+组成功的诱导出过敏性鼻炎的模型,WADm/m经致敏后未出现典型的过敏性鼻炎的模型,接种后5天WADm/m、WADm/+感染组与复合模型组窦腔感染均达到高峰,窦腔中白细胞集落和粘膜中白细胞数均明显高于对照组(P<0.05),10天后WADm/m、WADm/+小鼠单纯感染组窦腔感染逐渐减轻,14d后基本控制,而变态反应和感染复合模型组小鼠感染持续存在,与单纯感染组比较差异有显著性(P<0.05 ), 14d后复合模型组鼻灌洗液中仍培养出肺炎链球菌。结论采用肺炎链球菌ATCC 49619鼻内接种法成功地诱导出WADm/+小鼠急性鼻窦炎的模型中,肺炎链球菌在鼻腔鼻窦内的感染可被完全、自主、快速控制,但肥大细胞基因缺陷鼠wadm/m的鼻窦炎的模型,以及建立的wads的变态反应与鼻窦炎复合模型由于在鼻过敏基础上诱发鼻窦炎导致症状持续存在,并有演变成慢性炎症的倾向,提示变态反应在鼻窦炎的发病机制中具有关键性作用,肥大细胞缺失鼠未能成功的建立变应性鼻炎的模型,证实了肥大细胞在速发和迟发的变态反应中具有重要作用。肥大细胞基因缺陷鼠可作为研究鼻腔学病理生理学的工具.由于肥大细胞基因缺陷具有很好的实验对照,故该鼠可以作为研究鼻腔疾病的免疫学,病理学和生理学理想的动物。二、小鼠变应性鼻炎模型诱发细菌性鼻窦炎的研究目的探讨变应性鼻炎(allergic rhinitis,AR)动物模型诱发细菌性鼻窦的方法。材料与方法健康清洁级C57BL6/J小鼠40只,随机分为4组:A组(卵清蛋白+肺炎链球菌);B组(卵清蛋白+生理盐水);C组(PBS+肺炎链球菌);D组(PBS+生理盐水),每组各10只。①实验第0～8天A组和B组用10%卵清蛋白(ovalbumin)腹腔注射、第9～16天6%卵清蛋白鼻腔局部激发制成AR模型;C组和D组用PBS替代。②在AR基础上于实验第12天A组和C组鼻腔滴入肺炎链球菌ATCC 49619 (1.2×109CFU/ml);B组和D组用生理盐水代替。细菌滴鼻后5d处死动物,处死前各组动物内眦静脉采血,以间接酶联免疫吸附试验(ELISA)检测血清白细胞介素-5(IL-5)水平;取鼻面骨,石蜡包埋,连续切片,HE染色和甲苯胺蓝染色,计算机辅助光学显微镜观察肥大细胞浸润情况,计算每平方毫米鼻窦黏膜中多形核中性粒细胞(polymorphonuclear neutrophils,PMN)和嗜酸粒细胞(eosinophil,Eos)数。结果A组和B组分别有9只和8只动物AR建模成功,鼻部症状、黏膜水肿和微血管扩张明显,C组症状轻微,D组无炎症表现。A组鼻窦黏膜PMN密度为(139.29±26.51)mm2,高于B组(70.67±16.74)mm2、C组(63.04±14.73)mm2和D组(40.24±14.05)mm2 (P均<0.01); A组和B组Eos密度和IL-5水平分别为(134.63±25.49)mm2、(48.21±13.94)pg/ml和(116.21±25.17)mm2、(40.83±7.78)pg/ml,均高于C组(16.71±2.68)mm2、(23.89±8.65)pg/ml(P均<0.05)和D组(13.39±4.95)mm2、(24.58±6.50)pg/ml(P均<0.05)。结论鼻-鼻窦局部变应性炎症反应增加了鼻窦黏膜的细菌感染;单纯的变应性炎症不会引起鼻窦感染,必须要有致病菌的存在。三、肺表面活性物质相关蛋白D在鼻粘膜中的表达肺表面活性物质相关蛋白D是胶原凝集素家族的承运在气道固有免疫的防御反应中具有重要作用,在肺的炎症性疾病和感染性疾病,如哮喘,肺的疾病,囊性纤维化,SPD的表达及功能的改变具有重要的意义,本研究旨在探讨SPD在鼻腔是否存在,通过探讨小鼠慢性鼻窦炎模型中鼻黏膜表面活性蛋白D(SP-D)表达、以探讨肺表面活性物质相关蛋白在慢性鼻窦炎发病中的作用机制。方法1、动物组:动物模型的制备健康清洁级Wadm/m小鼠(A、C、E、G)和Wadm/+小鼠(B、D、F、H)各40只,每组随机分为4组:A组、B组(卵清蛋白+生理盐水);C组、D组(卵清蛋白+肺炎链球菌);E组F组(PBS+肺炎链球菌);G组、H组(PBS+生理盐水),每组各10只。①实验第0～8天A、B、C和D组用10%卵清蛋白(ovalbumin)腹腔注射、第9～16天6%卵清蛋白鼻腔局部激发制成AR模型;其余四组用PBS替代。②在AR基础上于实验第12天C、D、E、F组鼻腔滴入肺炎链球菌ATCC49619 (1.2×109CFU/ml);A、B组和G、H组用生理盐水代替。用石蜡包埋鼻面骨,进行连续切片,应用免疫组化法检测鼻粘膜中表面活性蛋白D(SP-D)的表达。2:临床资料:回顾性分析了我科2005年-2007年住院接受接受鼻内镜手术的患者鼻组织标本随机选取慢性单纯性鼻炎10例,鼻息肉组10例,有明确的过敏性鼻炎合并鼻息肉10例组织标本进行SPD的染色分析。结果鼠及人的鼻粘膜标本提示SPD位于鼻粘膜的呼吸上皮的表面,另外在粘膜下浆液性腺体及导管上皮均存在表达,在增生纤维组织中可见散在阳性表达的炎症性细胞。结论SPD在鼻粘膜存在表达,SPD可能参与鼻腔的固有免疫,外源性SP-D或人工重组的SP-D为治疗呼吸道的变应性疾病以及慢性炎症提供了新的思路,提高机体的固有免疫能力。更多还原

【Abstract】 Mast cell deficient (Ws/Ws) rats and wild-type controls(+/+) rats will be chosen to studythe role of mast cells in the pathogenesis of nasal allergy. Ws/Ws rats will be sensitized toovalbumin followed by development of bacterial rhinosinusitis model through intranasalinoculation of selected bacteria. Inflammatory cell infiltration, mast cell degranulation as wellas bacterial-epithelial cell interactions will be quantitatively measured by a 3-D methods. At thesame time, mitochondria and autophagosomes in the nasal epithelia will be studied throughelectron microscopy. In addition, epithelial function will be measured by short-circuit current（Isc）on the nasal epithelia and and by transepithelial flux of horserdish peroxidase (HRP).Micro-osmotic pump will be used for chemical interference. We believe that allergicinflammatory mucosal disease in the upper respiratory tract may be initiated firstly and mainlyby mast cell, instead of the current conception of Th1/Th2 imbalance. Mast cell-dependentallergic reaction is a key factor for inducing the bacteria rhinosinusitis.PartⅠ.Establishment of a mast cell-deficient mice Model of AcuteBacterial Rhinosinusitis by Allergic RhinitisObjective using to examine mast cell function in the nasal cavity，to explore the infectivecourse of bacterial rhinosinusitis based on allergic rhinitis in homozygous mutants Wadsm/m andheterozygote Wadsm/+（The mutation localized at W/c-kit）and the difference between themafter intranasal streptococcus pneumoniae inoculation. Methods Respectively 40 mice ofWadsm/m and Wadsm/+ were randomly divided averagely into 4 groups: A、B (OVA+NS) C、D(OVA+S.p.),E、F(PBS+ S.p.) and G、H(PBS+NS).①Group A、B、C and D were sensitizedto OVA（10％）by intraperitoneal injection on days 0 through 8, and exposed to OVA(6%)intranasally on days 9 through 16, as to induce allergic inflammation. PBS was used in group Cand D in the same way.②Subsequently, group C、D、E and F were inoculated S.p.（1.2×109CFU/ml）intranasally on day 12, and NS was used in other groups .After 2, 5, 10 and14 days, Blood was gathered from the orbital venous sinus after anesthesia. nasal lavage cuhureswere obtained and then the mice were killed. On the 6th day after inoculation, mice were killed.The heads were embedded with paraffin and serial sections were made and stained withhematoxlin-eosin and toluidine blue for histological analysis. The number of polymorphounclearneutrophils and eosinophils per square millimeter of sinus mucosa (PMN/mm2 and Eos/ mm2)were calculated by the use of computer aided microscope. ed. The heads were embedded with paraffin and serial sections were made for histological analysis. The percentage of sinus cavityoccupied by neutrophil cluster (% cluster) and the number of polymorphonuclear leukocytes persquare millimeter of sinus mucosa (PMN/ mm2) were calculated by the use of a computer aidedmicroscope in conjunction with a reconstruction and image analysis system. Results % Clusterand PMN/ mm2 in infected mice appeared to peak on five days，which were significantlyheavier than those in controls( P < 0. 05) . The infection in simple infection decreased by twoweeks. But in complex model of allergy and infection contrast to simple infection, the infectionin wadm/m had not been controlled and Streptococcus pneumoniae were still seen in the nasallavage cuhure by two weeks. Conclusion:AR models were successfully established from wadm/+mice . It was found that mast cell-deficient mice didn`t exhibited .Acute bacterial rhinosinusitisin wads are successfully induced by intranasal inoculation of streptococcus pneumoniae. Thisbacterial infection in the group of simple infection controlled completely and rapidly. Our datademonstrate that an ongoing local allergic response augments bacterial infection in these mice.Incontrast failed to control rhinosinusitis and had a tendency to chronic inflammation, suggestingthat mast cell was important for clearance of bacteria from rhinosinus and mast cell-deficientWads was a convenient tool for investigation of the pathogenesis of experimental rhinosinusitis.Mast cell-deficient mice has been developed for study of a variety of pathological,immunological, and physiological processes in the respiratory tract ,beacause this is a veryconvenient site for experimental manipulations.PartⅡ.Establishment of a Mouse Model of Acute Bacterial Rhinosinusitis by AllergicRhinitisObjective To develop a animal model of bacterial rhinosinusitis in mice with allergicrhinitis, and explore whether ongoing allergic rhinitis enhances the acute sinus infection andinflammation associated with Streptococcus pneumoniae. Methods 40 mice of C57BL6/J wererandomly divided averagely into 4 groups: A (OVA+S.p.), B(OVA+NS), C(PBS+ S.p.) andD(PBS+NS).①Group A and B were sensitized to OVA（10％）by intraperitoneal injectionon days 0 through 8, and exposed to OVA(6%) intranasally on days 9 through 16, as to induceallergic inflammation. PBS was used in group C and D in the same way.②Subsequently, groupA and C were inoculated S.p.（1.2×109CFU/ml）intranasally on day 12, and NS was used ingroup B and D. On the 6th day after inoculation, mice were killed. Blood was gathered from theorbital venous sinus after anesthesia. The heads were embedded with paraffin and serialsections were made and stained with hematoxlin-eosin and toluidine blue for histologicalanalysis. The number of polymorphounclear neutrophils and eosinophils per square millimeterof sinus mucosa (PMN/mm2 and Eos/ mm2) were calculated by the use of computer aided microscope. Results AR models were successfully established on 9 from group A and 8 fromgroup B. Histological examination of the sinus of group A and B revealed significant mucosaledema and dilated venules. The symptoms were mild in group C, and no symptom wasobserved in group D. PMN/mm2 in group A（139.29±26.51）mm2, were significantly heavierthan group B（70.67±16.74）mm2, C（63.04±14.73）mm2 and D（40.24±14.05）mm2 respectively（P＜0.01）; Eos/ mm2 and serous IL-5 level in group A（134.63±25.49）mm2,（48.21±13.94）pg/ml and B（116.21±25.17）mm2、（40.83±7.78）pg/ml, were higher than group C（16.71±2.68）mm2,（23.89±8.65）pg/ml（P＜0.05）and D（13.39±4.95）mm2,（24.58±6.50）pg/ml（P＜0.05）. Conclusion Our data demonstrate that an ongoing local allergic response augmentsbacterial infection in these mice, and allergic sensitization alone does not augment the sinusinfection without pathogenic bacterium.Pa r tⅢ.Sur f a c t ant Pr o t e in D Expr e s s i on in Chr oni c Rhino s inus i t i s andImmune ResponsesObjectives Surfactant-associated proteins (SP) D are in the family of collectin proteinsthat play an integral part in the innate defense system. SP-D expression and function are alteredin a variety of inflammatory and infectious diseases of the lungs, such as asthma, allergies, andcystic fibrosis. Our studies are the first to identify the presence of these proteins in the nasalcavity. The objective of this study was to immunolocalize SP-D in human sinonasaltissue.Methods Respectively 40 mice of Wadsm/m and Wadsm/+ were randomly dividedaveragely into 4 groups: A、B (OVA+NS) C、D(OVA+S.p.),E、F(PBS+ S.p.) and G、H(PBS+NS).①Group A、B、C and D were sensitized to OVA（10％）by intraperitoneal injection on days0 through 8, and exposed to OVA(6%) intranasally on days 9 through 16, as to induce allergicinflammation. PBS was used in group C and D in the same way.②Subsequently, group C、D、Eand F were inoculated S.p.（1.2×109CFU/ml）intranasally on day 12, and NS was used in othergroups . After 2, 5, 10 and 14 days, The heads were embedded with paraffin and serial sectionswere made for histological analysis. nasal mucosal biopsies were performed in patients withvarious forms of chronic hyperplastic rhinosinusitis with nasal polyposis and nondiseasedmucosa from patients undergoing (n = 10) Immunolocalization of surfactant proteins wasperformed with antibodies SP-D using immunoperoxidase staining technique. Isotype-negativecontrols were performed on all specimens.RESULTS: Analyses of mucosal biopsy specimensfrom nasal tissue reveals staining within respiratory and intermediate (metaplastic)-type surfaceepithelium. In addition, staining was intense in the submucosal ductal epithelium of theseromucinous glands. These properties appear to be consistent regardless of disease state andlocation within the sinuses.CONCLUSION: These are secreted proteins that are intricately involved in innate immunity in the lungs. Their secretion in the upper airway indicates thatfuture studies may allow manipulation of these proteins and development of novel treatments forsinonasal pathology.更多还原