【作者】 刘娜；

【导师】 林勇；

【作者基本信息】 东南大学 ， 临床医学， 2015， 硕士

【摘要】 [目的]探讨胃食管反流性疾病(gastroesophageal reflux disease, GERD)豚鼠模型肺及延髓中神经激肽-1受体(neurokinin-1 receptor, NK-1R)的表达及与豚鼠咳嗽反射敏感性(cough reflex sensitivity, CRS)之间的关系。[材料与方法]取CRS正常的健康雄性Hartly豚鼠随机分为五组：空白组,生理盐水组,模型组,模型+肉毒毒素-A(botulinum toxin-A, BTX-A)组,模型+生理盐水组。模型组豚鼠食管下端灌注含0.5%胃蛋白酶的盐酸溶液(pH=0.86),7-8滴/min,20min/天,连续15天。食管下端酸性溶液灌注联合气道BTX-A注射构建模型+BTX-A组。造模完成后再次测量豚鼠CRS,并通过免疫组织化学染色法检测肺组织NK-1R的表达,免疫组织化学荧光染色法及Westernblot法检测脑组织延髓中NK-1R的表达改变。[结果](1)食管下端多次酸性溶液灌注建立的GERD模型组豚鼠与对照组相比,其咳嗽次数增加(51.17±10.19 vs 29.50±13.40,51.17±10.19 vs 26.80±9.83,P<0.05),潜伏时间缩短(8.24±4.73 vs 38.17±9.91,8.24±4.73 vs 31.92±4.78,P<0.05),即气道CRS增高。模型+BTX-A组较模型组咳嗽次数减少(23.33±7.53 vs 51.17±10.19,P<0.05),潜伏时间未见明显改变(11.76±3.29 vs 8.24±4.73,P>0.05)。(2)免疫组织化学染色实验结果示肺组织及脑内延髓迷走复合体(dorsal vagal complex, DVC)中有NK-1R表达改变。模型组肺组织及延髓DVC中NK-1R相较于空白组及生理盐水组,其免疫阳性物质表达增多(108.48±25.71 vs 38.54±17.62,4.99±0.44 vs 1.96±0.16,P<0.05),反应增强。模型+BTX-A组与模型组相比,肺组织及DVC内NK-1R表达明显下降(21.59±13.45 vs 108.48±25.71,1.32±0.11 vs 4.99±0.44,P<0.05)。(3) Western blot结果显示模型组延髓NK-1R蛋白表达相较于空白及生理盐水组,其相对表达量增加(1.00±0.43 vs 0.46±0.34,1.00±0.43 vs 0.50±0.37,P<0.05),气道注射BTX-A后与模型组相比,NK-1R蛋白相对表达量下降(0.49±0.33 vs 1.00±0.43,P<0.05)。[结论]多次食管下端酸性溶液灌注建立的豚鼠GERD模型其气道CRS增高,表现为咳嗽次数增加,咳嗽潜伏时间缩短。GERD豚鼠模型肺组织及延髓DVC内NK-1R的表达增加可能与神经源性炎症的产生相关,增加的NK-1R可能参与豚鼠气道CRS增高的调节机制。更多还原

【Abstract】 [Objection] To explore the relationships between cough reflex sensitivity and the neurokinin-1 receptor expression in lung and dorsal vagal complex of gastroesophageal reflux model.[Methods] Healthy male Hartly guinea pigs that cough reflex sensitivity are normal were randomized into five groups, control group, physiological saline group, model group, model+BTX-A group and model+NS group. Use the method that esophageal intubation and perfused HC1 continuous 15 days to establish a guinea pig model of GERD. Use or not use botulinum toxin-A, measured citric acid-induced cough times and latency in the end of building model and observed neurokinin-1 receptor expressions in the lung and dorsal vagal complex by immunohistochemical staining and/or western blot.[Results] (1) Compared with the control groups, the cough times of model group was significantly increased and the cough latency significantly shorter (51.17±10.19 vs29.50± 13.40,8.24±4.73 vs 38.17±9.91, P<0.05). Injectd botulinum toxin-A into airway, the cough times were significantly decreased(23.33±7.53 vs 51.17±10.19, P<0.05), but the cough latency was no significant difference (11.76±3.29 vs 8.24±4.73, P>0.05) compared with the model group. (2) The neurokinin-1 receptor immunohistochemical staining results from the lung tissue and dorsal vagal complex suggested that the numbers of neurokinin-1 receptor-immunoreactive substances were significantly increased, with a stronger response in the model group compared with the control groups(108.48±25.71 vs38.54±17.62,4.99±0.44 vs1.96±0.16, P<0.05). After used the botulinum toxin-A, neurokinin-1 receptor-immunoreactive substances were decreased significantly compared with model group(21.59±13.45 vs 108.48±25.71,1.32±0.11vs4.99± 0.44, P<0.05). (3) In the model groups, the expression of neurokinin-1 receptor protein in dorsal vagal complex was increased compared with control groups (1.00±0.43 vs 0.46±0.34, P<0.05). Pretreated botulinum toxin-A, the expression of ncurokinin-1 receptor protein lever was decreased significantly compare with model group (0.49±0.33 vs 1.00±0.43, P<0.05).[Conclusion]Increased of cough time and shortened cough latency suggested that GERD inhanced the cough reflex sensitivity in guinea pigs and it can be blocking by injectable botulinum toxin-A into airway. And the expression of neurokinin-1 receptor may play an important role in the gastroesophageal reflux model of guinea pigs with the increase of cough reflex sensitivity.更多还原