凝血酶对哮喘大鼠气道炎症作用的研究

发布时间：2018-08-09 18:07

【摘要】：目的支气管哮喘是儿童时期的常见呼吸道疾病。支气管哮喘会引起反复发作的喘息、气急、胸闷或咳嗽等症状,给病患带来了极大的痛苦和沉重的经济负担。IL-6是参与哮喘发生的一种炎症因子,在哮喘气道炎症中具有重要作用。近年研究证明：凝血酶是参与呼吸道炎症的介质,可以介导前炎性转录因子NF-κB的活化。本实验外源性给予凝血酶及凝血酶抑制剂,建立标准大鼠哮喘动物模型。通过①ELISA法检测各组大鼠肺泡灌洗液(BALF)中凝血酶活性；②HE染色观察各组大鼠肺组织中炎症细胞浸润情况；③免疫组化法检测各组大鼠肺组织中白介素(IL-6)蛋白表达水平；④western blot法检测各组大鼠肺组织中核因子-κB(Nuclear factor kappa B, NF-κB)蛋白表达情况。探讨凝血酶对哮喘大鼠气道炎症的影响,从而为支气管哮喘气道炎症发生机制的探讨提供新的思路。方法将24只成年雌性大鼠随机分为4组,正常对照组(N组)、哮喘模型组(A组)凝血酶组(T组)、凝血酶抑制剂组(H组),每组6只。以卵蛋白(OVA)致敏并吸入激发制备大鼠哮喘模型。实验组(A组、T组、H组)大鼠分别于第1、8天每只大腿内侧皮下注射OVA悬液1ml (OVA10mg+氢氧化铝200mg+0.9%生理盐水至1ml),同时腹腔内注射灭活百日咳杆菌悬液1ml (约6×109个菌株)作为佐剂。第15天始使用402超声雾化器(上海四菱医疗器械厂)向置于密闭有机玻璃容器内的大鼠雾化吸入2%卵蛋白(现配)每次30分钟,每周3次,连续激发6周。每次雾化前30min,T组每只大鼠给予雾化吸入凝血酶72u、H组每只大鼠给予雾化吸入凝血酶抑制剂-水蛭素500u/kg。正常对照组所有干预均使用生理盐水代替OVA。造模成功后,取大鼠肺组织和BALF。采用ELISA法测定BALF中凝血酶活性, HE染色观察肺组织炎症浸润情况,免疫组化方法检测肺组织中IL-6表达,western blot法测定各组大鼠肺组织中NF-κB蛋白表达情况。结果 1.一般情况观察实验组(A组、T组、H组)大鼠表现为毛发失去光泽,在造模激发过程中出现不同程度的咳嗽、打喷嚏、呼吸急促、腹肌紧张、四肢瘫软、行动迟缓、反应迟钝等症状。正常对照组则无上述症状。 2.凝血酶活性哮喘组BALF中凝血酶活性高于正常对照组(P0.05)；凝血酶组BALF中凝血酶活性高于哮喘组(P0.05)；凝血酶抑制剂组凝血酶活性低于凝血酶组(P0.05)。 3.观察肺组织炎症浸润情况 HE染色显示正常对照组气道无炎症浸润,哮喘组气道有炎症细胞的浸润,凝血酶组有大量炎症细胞浸润,较哮喘组明显,凝血酶抑制剂组炎症细胞浸润较凝血酶组减轻。 4.免疫组织化学法检测肺组织中IL-6的表达正常对照组免疫组化显示肺组织IL-6基本无表达；哮喘组IL-6表达较正常对照组增多；凝血酶组IL-6表达最多,较哮喘组高；凝血酶抑制剂组IL-6表达较凝血酶组明显减少。 5.肺组织中NF-κB蛋白表达情况采用western blot方法对NF-κB蛋白的表达情况进行检测,结果显示正常对照组NF-κB微弱表达,哮喘组NF-κB表达较正常对照组升高,凝血酶组NF-κB表达水平最高,凝血酶抑制剂组表达水平较凝血酶组低。结论 1.大鼠支气管哮喘模型建立成功。 2.哮喘大鼠气道凝血酶活性升高,凝血酶可促进哮喘大鼠气道炎性细胞浸润。 3.凝血酶可促进哮喘大鼠气道NF-κB的表达,进而促进炎性因子工L-6的表达,从而参与哮喘炎症的发生。
[Abstract]:objective
Bronchial asthma is a common respiratory disease in childhood. Asthma can cause recurrent symptoms such as wheezing, air urgency, chest tightness, or coughing, which bring great pain and heavy financial burden to the patients..IL-6 is an inflammatory factor involved in asthma and plays an important role in the airway inflammation of asthma. Thrombin, a medium for respiratory inflammation, can mediate the activation of NF- kappa B of the proinflammatory transcription factor. In this experiment, thrombin and thrombin inhibitors were given to establish a standard rat model of asthma. The activity of thrombin in alveolar lavage fluid (BALF) was detected by ELISA. (2) the lung group of rats in each group was observed by HE staining. The expression of interleukin (IL-6) protein in lung tissues of each group was detected by immunohistochemistry; (4) the expression of nuclear factor kappa B (Nuclear factor kappa B, NF- kappa B) in lung tissues of rats in each group was detected by immunohistochemical method. The effect of thrombin on airway inflammation in asthmatic rats was investigated. To explore the pathogenesis of airway inflammation in asthma provides new ideas.
Method
24 adult female rats were randomly divided into 4 groups, the normal control group (group N), the asthma model group (group A) thrombin group (group T), the thrombin inhibitor group (group H), 6 rats in each group. The rat model of asthma was prepared with ovalbumin (OVA) sensitization and inhalation excitation. The experimental group (group A, T group, H group) was subcutaneously injected with OVA suspension 1ml on the inside of each thigh on day 1,8, respectively. (OVA10mg+ aluminum hydroxide 200mg+0.9% to 1ml), and intraperitoneal injection of pertussis pertussis suspension 1ml (about 6 x 109 strains) as adjuvant. The 402 ultrasonic nebulizer (Shanghai four medical device factory) was used to atomization of rats in a closed plexiglass container for 30 minutes each time and 3 per week for fifteenth days. Every rat in group T was given inhalation of thrombin 72u for 6 weeks. Each rats in group T were given inhalation of thrombin 72u before each atomization. Each rat in group H was given inhalation of thrombin inhibitor - hirudin 500u/kg. in the normal control group. All the rats were treated with normal saline instead of OVA.. The rat lung tissue and BALF. were taken to determine the thrombin activity in BALF by ELISA method. The inflammatory infiltration of lung tissue was observed by HE staining. The expression of IL-6 in lung tissue was detected by immunohistochemical method, and the expression of NF- kappa B protein in lung tissues of each group was measured by Western blot method.
Result
1. general situation observation
The rats in the experimental group (group A, group T, group H) showed the loss of luster in the rat hair. In the process of stimulating the model, there were different degrees of cough, sneezing, shortness of breath, abdominal muscle tension, limp limbs, slow action, slow reaction and so on. The normal control group had no symptoms.
2. thrombin activity
The thrombin activity in the BALF of the asthmatic group was higher than that in the normal control group (P0.05), and the thrombin activity in the thrombin group was higher than that in the asthma group (P0.05), and the thrombin activity in the thrombin inhibitor group was lower than that of the thrombin group (P0.05).
3. observation of inflammatory infiltration of lung tissue
HE staining showed no inflammatory infiltration in the airway of the normal control group. There were inflammatory cells in the airway of the asthmatic group, and a large number of inflammatory cells were infiltrated in the thrombin group. The inflammatory cell infiltration in the thrombin inhibitor group was less obvious than that in the asthma group.
4. Detection of IL-6 expression in lung tissue by immunohistochemistry
In the normal control group, the expression of IL-6 in the lung tissue was not expressed in the normal control group; the expression of IL-6 in the asthma group was more than that in the normal control group; the expression of IL-6 in the thrombin group was most, higher than that in the asthma group, and the expression of IL-6 in the thrombin inhibitor group was significantly lower than that in the thrombin group.
The expression of NF- kappa B protein in 5. lung tissues was detected by Western blot method. The results showed that the expression of NF- kappa B in the normal control group was weak, the expression of NF- kappa B in the asthmatic group was higher than that in the normal control group. The expression level of NF- kappa B in the thrombin group was the highest, and the expression level of the thrombin inhibitor group was lower than that of the thrombin group.
conclusion
The 1. rat model of bronchial asthma was established successfully.
2. The airway thrombin activity in asthmatic rats increased, and thrombin could promote the airway inflammatory cell infiltration in asthmatic rats.
3. Thrombin can promote the expression of NF-kappa B in the airway of asthmatic rats, and then promote the expression of inflammatory factor L-6, thus participate in the occurrence of asthmatic inflammation.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R725.6

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