【作者】 李芹子；

【导师】 孔灵菲；

【作者基本信息】 中国医科大学 ， 内科学， 2009， 博士

【摘要】 近年来,胃食管反流性疾病（gestroesophageal reflux disease,GERD）与支气管哮喘的关系一直是共同关注的焦点,随着有效的抑酸药物和便携式pH测试仪的出现,逐渐意识到胃食管反流（gestroesophageal reflux,GER）GER可能是哮喘的一个激发因素。为探讨GERD与哮喘的关系,合适的动物模型的建立是不可缺少的手段之一。目前认为GER性呼吸系统疾病的主要原因是食管-支气管反射和神经源性炎症,即由于盐酸刺激食管内的酸敏感受体促发迷走神经活动产生的,GER通过该反射引起气道高反应。在酸性胃食管反流状态下,由于食管-支气管反射,气道感觉神经受到刺激,一方面,通过轴突反射释放神经肽,引起神经源性炎症;另一方面,冲动传至中枢,与孤束核及相应节段脊髓背角的神经元形成突触,引起呼吸反射。研究发现该反射至少部分通过迷走神经介导,反射弧的感受器分布在气道上皮的感觉神经末梢,它们的胞体位于结状神经节和上段脊神经节（C₇-T₅）。P物质（substance P,SP）作为神经肽家族中最重要的成员,其发挥多种生物学效应是通过与其高亲和力受体NK-1的结合来完成的。研究显示,SP参与了哮喘和酸灌注食管的病理过程,但SP是否参与了胃食管反流性气道高反应的发病过程以及食管灌注盐酸豚鼠模型的C₇-T₅脊神经节和相应节段脊髓背角内的SP是否有变化目前尚不清楚。神经生长因子（nerve growth factor,NGF）是哮喘发病过程中的一个重要介质,支气管哮喘动物模型的研究结果显示NGF能引起气道高反应,但NGF是否参与GER性哮喘的发病机制目前尚不清楚。故以多次盐酸灌注豚鼠食管动物模型为研究对象,探讨SP在呼吸道和内脏传入部位的表达和NGF在肺组织中的表达。进一步研究食管-支气管反射及其相关的神经源性炎症的作用,试图为食管一支气管反射找出明确的解剖学证据。1、多次盐酸灌注豚鼠食管气道高反应动物模型的建立普通级健康雄性白化豚鼠30只,体重350g—450g,随机分为3组:A组:PBS对照组;B组:HCL模型组;C组:盐酸灌注+SR140333干预组。食管酸灌注法:盐酸氯胺酮注射液50g/L腹腔注射（1mL/kg）轻度麻醉豚鼠,仰卧固定于手术台,经口插5F胃管入食管的中、下段,以8滴/min速率缓慢灌注0.1mmol/L HCl（含0.5%胃蛋白酶）溶液,每次20min,每天一次,连续14d。对照组:用PBS代替盐酸灌注食管,方法同上。拮抗剂用法:将SR140333溶于1%DMSO的蒸馏水中,配成0.5mg/ml,按1mg/kg在每次灌酸前30分钟进行腹腔注射。2、气道反应性测定所有豚鼠于末次灌注后24h行气道反应性测定,戊巴比妥腹腔注射麻醉,将三组豚鼠应用AniRes2005实验动物肺功能检测分析系统（北京贝兰博科技有限公司）检测气道阻力。3、取支气管肺泡灌洗液（bronchoalveolar lavage fluid,BALA）,BALF细胞成分计数;血管灌注,豚鼠处死,取豚鼠食管,气管,肺组织,C₇-T₅段脊髓和相应节段脊神经节。4、HE染色观察食管,气管和肺组织病理变化将豚鼠食管、气管和肺组织OCT包埋、切片后,进行HE染色,光镜下观察并照相。5、免疫组织化学染色检测SP和NGF的表达取对照组、实验组、干预组的气管、肺组织、C₇-T₅段脊髓和相应节段脊神经节切片,参照二步法免疫组织化学试剂盒步骤测定各部位SP和NGF的表达情况。6、RT-PCR检测肺组织、C₇-T₅段脊髓SP的表达,图像分析。7、免疫印迹检测肺组织中NGF的表达肺组织中蛋白提取和定量后,取等量样品进行聚丙烯凝胶电泳,经一抗和二抗孵育后显色,图像分析。8、统计学分析所有原始数据采用SPSS11.5统计软件进行数据分析。所有结果均表示为（?）±s,样本均数比较采用单因素方差分析方法,正态分布及方差齐时,两两比较采用LSD检验;非正态分布采用轶和检验,p＜0.05为差异有统计学意义实验结果1、三组豚鼠食管、气管和肺组织病理检测食管病理学HE染色显示,可见正常对照组豚鼠食管结构基本正常;模型组豚鼠食管上皮层不同程度增厚,以基底层及刺层细胞增生为主,上皮角化过度,乳头延长;固有层中性粒细胞、嗜酸性粒细胞、巨噬细胞和淋巴细胞浸润;粘膜下层内血管扩张,充血,符合轻度食管炎改变。支气管肺组织病理学HE染色显示,对照组豚鼠支气管肺组织结构基本正常;模型组气管可见纤毛柱状上皮明显增生,部分上皮脱落,粘膜和粘膜下层血管充血、扩张,腺体增生、肥大,杯状细胞增多,炎症细胞如巨噬细胞、中性粒细胞、嗜酸性粒细胞和淋巴细胞浸润,肺组织、细支气管管壁及管腔内可见大量淋巴细胞及嗜酸性粒细胞浸润,细支气管管壁增厚,管腔狭窄,部分可见粘液栓。SR140333干预组模型的病理变化均较实验组有所减轻。2、气道反应性测定随着乙酰胆碱浓度的成倍递增,模型组与对照组的呼气阻力均有增加,当浓度到达25μg/kg体重以上时,模型组与对照组比较有显著差异（P＜0.01）。给予SR140333干预后,呼气阻力较实验组明显改善。3、BALF中白细胞计数及分类模型组BALF中细胞总数及嗜酸细胞百分比均高于对照组（P＜0.01）。给予SR140333干预后,与模型组比较明显减少。4、免疫组织化学染色及分析正常对照组、模型组和SR140333干预组支气管、肺组织、C₇-T₅段脊神经节和相应节段脊髓背角中均有SP的表达,但强弱不同,模型组明显高于其他两组（P＜0.01）。模型组下呼吸道NGF的表达明显高于正常对照组（P＜0.01）。5、RT-PCR检测SP的表达各组肺组织和C₇-T₅段脊髓背角中SP含量不同,模型组高于正常对照组、SR140333抗体组（P＜0.01）。6、免疫印迹检测NGF的表达模型组和对照组NGF蛋白含量不同,模型组高于对照组。结论1、成功建立了多次盐酸灌注豚鼠食管气道高反应动物模型,为进一步探讨GERD与哮喘的关系提供了帮助。2、SP在豚鼠下呼吸道、C₇-T₅段脊模型较对照组明显增加。提示胃食管反流性气道高反应存在气道神经源性炎症,并且神经源性炎症参与了胃食管反流性气道高反应的中枢神经的发病机制。3、NK-1受体拮抗剂可以降低多次盐酸灌注豚鼠食管气道高反应动物模型的气道阻力。NK-1受体拮抗剂可以下调多次盐酸灌注豚鼠食管气道高反应动物模型下呼吸道和内脏传入部位SP的表达,即阻断了其气道神经源性炎症。4、NGF在豚鼠下呼吸道有表达,NGF在GER性气道高反应豚鼠下呼吸道中表达明显,提示NGF可能参与了GER性哮喘的发病。更多还原

【Abstract】 AIMGastro-oesophageal reflux disease（GERD） is believed to lead to extra-oesophageal symptoms and complications,primarily in the respiratory tract,such as asthma.With appearance of the drug of effective acid-inhibitor and the 24h PH-monitoring,that GER might be an excitation factor of asthma is recognized gradually.To discuss the relationship between the GER and asthma,appropriate animal model is necessary.The main reason of GER in respiratory tract is the oesophageal-bronchial reflux,that is the acid sensitive receptors in the oesophagus are stimulated by acid which prime the vagus nerve,the airway hyperresponsiveness is caused by this reflux.Sensory nerve in the airway is stimulated because of the oesophageal-bronchial reflux,releases neuropeptide through axon-reflux,causes neurogenic inflammation.On the other hand,impulse conducts to the CNS,forms synaptic with nTS and correspondent spinal dorsal horn,causes respiratory reflux which induces by vagus nerve partly.The receptors of the reflex arc innervate in the airway epithelium and its bady locates in the vagal nodose ganglia and the vagal jugular ganglia.Substance P acts as an important role of neuropeptide family,the diverse actions of SP are often fulfilled by binding to its high-affinity specific receptor,the NK-1.SP is confirmed to participate in the pathogenesis of asthma and acid perfusion of the esophagus,but whether SP also takes part in the process of GER inducing hyperresponsiveness and whether it expresses in the spinal ganglia and corresponding spinal dorsal horn remain open questions.Nerve growth factor,（NGF） is an important medium in pathogenesis of asthma,but whether it take part in the pathogenesis of GER inducing hyperresponsiveness is unknowed.In this study,we detected the expression of SP and NGF in the airway and viscerosensory afferent sites in the external repetitive acid-perfusion oesphageal guinea pig model to test the oesophagus - branchus reflex and neuroginic inflammation.Material and methods1、AnimalsSpecific pathogen-free albino guinea pigs weighing 350-450 g of both sexes were provided by the Laboratory Animal Center,College of Basic Medicine,China Medical University.Thirty guinea pigs were randomized into 3 groups:A group:namely the phosphate buffer solution（PBS）control（n=10）;B group:experimental group（n=10）;C group:SR140333 group（n=10）.In the experimental group the oesophagi were challenged with a solution of hydrochloric acid（HCI） containing 1 g/L pepsin（HCl-P; pH 1.0）.In the control group phosphate buffered saline（PBS）（pH 7.0） was used to treat the oesophagi.HCl was used in a 0.1 N solution,the same concentration found in the human stomach.Pepsin was added to simulate the gastric contents during the digestive process.In the HCl-P group,on the day of experimentation,guinea pigs were maintained under ketamine anesthesia delivered intraperitoneally at 50 mg/kg,with additional injections given as necessary.Animals were placed in supine position,and a 5-Fr catheter was inserted through the mouth and into the lumen of the middle and lower oesophagus.The oesophagus of each animal was perfused with HCl-P for 20 min/d for 14 d at a flow rate of 0.3 ml/min via a recirculating system.In the control group,animals received an oesophageal perfusion of PBS.During perfusion,all animals were in a 30°anti-Trendelenburg position.The guinea pigs in C group were injected pertioneally with ST 140333 30min before acid perfusion.2、Determination of airway hypersensitivityThe determination of airway responsiveness to acetylcholine（ACh） was measured 24 hr after the last perfusion,pentobarbital anesthesia.Measure the airway hypersensitivity with AniRes 2005 animal lungs function analysis（Beijing Beilanbo Technology Co.Ltd.）3、Vascular perfusion,sacrifice of guinea pigs,sample preparationCell numbers in BALF were detected;pathological changes of the lower oesophagus,bronchial and lung tissues of different groups were detected by HE staining.4、Hematoxylin-Eosin（HE）staining of the lower oesophagus, bronchial and lung tissues pathologyThe lower oesophagus,right main bronchus,and right lung tissues were removed and fixed in 4%polyformaldehyde for subsequent frozen section HE staining to observe the pathological changes using light microscopy（Olympus BX51,Japan）.5、Detecting the expression of SP by means of immunohistochemistry technique in lower respiratory tract and viscerosensory afferent sites of the guinea pigs among different groups.SP protein was observed in groups of A B C,referencing to two-step immunohistochemistrical kits.6、The mRNA levels of SP in the lung and spinal dorsal horn were analyzed by RT-PCR,withβ-actin as an internal control..7、Western blot method to detect protein level of NGF in the lung.After extraction and quantification of protein from the lung,the samples were electrophoresed with SDS-PAGE.The samples were culture with primary and secondary antibody and detected.8、Statistical AnalysisThe data were analyzed with SPSS 13.0 software.The results are expressed as mean values±standard deviation.Differences between the groups were analyzed by t-test and one-way analysis of variance（ANOVA） followed by Fisher’s LSD test.A P value of＜0.05 was considered statistically significant.Results1.Pathological changes of the lower oesophagus,bronchial and lung tissuesNo pathological changes of the oesophagus were observed in the PBS control animals.In the oesophagi of HCl-P-perfused animals,we observed infiltration of the mucosa by inflammatory cells,basal cell and spinous cell hyperproliferation,papillae hypertrophy,epithelia hyperkeratinization,squamous cell expansion,and increases in the number of fibrin cells.These results were consistent with the typical histological findings associated with low-grade reflux oesophagitis.Guinea pigs in the PBS control group presented with normal tracheal structures.In the HCl-P-treated model group, obvious hyperplasia,duplicated layer array,and nucleolus darkening of the ciliated columnar epithelium were observed.We observed large numbers of inflammatory cells, namely lymphocytes and eosinophils,infiltrating the submucosa.Airway epithelial desquamation was also evident in experimental animals.We also observed goblet cell metaplasia,ciliated columnar epithelium necrosis and degeneration,alveolus epithelium hyperproliferation,as well as in capillary vessels.Mucus plugs were seen in some of the bronchioles.Constitution of SR140333 group is releaser than that of the HCl-P-perfused group.2.The measurement of airway hyperresponsivenessIn response to increasing doses of intravenously administered ACh,all experimental guinea pigs showed dose-dependent increases in Re.However,when the dose of ACh was increased to 25 g/kg,the airway responsiveness increased significantly in HCl-P model animals when compared with PBS control animals. Airway resistance is more lessened in SR140333 group than the HCl-P-perfused.3.Cell numbers and types observed in guinea pig BALFThe total cell number,as well as lymphocyte and eosinophil counts were significantly greater in the BALF of HCl-P model animals than that of the PBS control animals（P＜0.01）.The total cell number and eosinophil is more lessened in SR140333 group than the HCl-P-perfused.4.Immunohistochemistry analysis for SPSP in the lung tissues,C₇-T₅ spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups（P＜0.01）.5.The mRNA expression of SPThe ratios of mRNA/β-actin mRNA in HCL-P model animals were increased significantly compared with control group and SR140333 group.6.Detectiong the expression of NGF in lung with Western blot experimentWestern blotting revealed significantly more intense expression of NGF in the lung from model guinea pigs than in the control（P＜0.01）.Conclusion1.A novel external oesophageal perfusion model to induce oesophageal,tracheal and pneumonic histological injury similar to that associated with gastroesophageal reflux（GER） was evaluated successfully.2.SP in the lung tissues,C₇-T₅ spinal ganglion and corresponding spinal dorsal horn was expressed in the three groups repectively,but their strengths were different, HCl-P-perfused group was significantly higher than the other two groups,which suggested that the neurognic inflammation of airway participated in the pathogenesis in the centre nerve of GER.3.NK-1 receptor antagonist may reduce the airway resistance in external oesophageal perfusion guinea pigs for reflux-associated respiratory symptoms.NK-1 receptor antagonist may also reduce the SP expression in the in lower respiratory tract and viscerosensory afferent sites of HCl-P-perfused group,which suggested that it restrained the neurognic inflammation of airway.4.NGF expression in the lung of control and model groups,and found increased NGF expression in the lung of model,suggest that NGF participates in the pathogenesis of GER inducing asthma.更多还原