【作者】 李英；

【导师】 王孟清；

【作者基本信息】 湖南中医药大学 ， 中医儿科学， 2012， 博士

【摘要】 目的：本文运用分子生物学、显微形态学、免疫学、病毒学等实验技术,研究咳喘宁口服液对呼吸道合胞病毒诱发哮喘模型大鼠STAT6蛋白及其mRNA的表达的影响,为进一步阐明咳喘宁防治病毒诱发儿童哮喘的作用机理提供依据。方法：①建立卵白蛋白哮喘大鼠模型并以呼吸道合胞病毒激发哮喘；②取支气管肺泡灌洗液(BALF)作细胞分类计数及比较BALF中IL-4与IL-12水平；分析IL-4、IL-12与STAT6蛋白和STAT6mRNA的相关性。采用免疫组织化学法和原位杂交法分别检测哮喘模型大鼠支气管中STAT6蛋白和STAT6mRNA的表达；③造模完成当天,常规分离PBS对照组、模型组各10只动物外周血单个核细胞,磁珠结合法分离CD4+T、CD8+T细胞,分别进行CD4+T、CD8+T细胞纯度鉴定与细胞活力检测,采用免疫荧光法,根据藻红蛋白(PE)荧光标记的抗体被激发出的荧光量多少,使用流式细胞仪确定T细胞内STAT6蛋白的表达水平,以中药咳喘宁进行干预,动态观察细胞培养过程中,咳喘宁对CD4+T、CD8+T细胞中STAT6蛋白表达的影响。结果：与单纯OVA致敏哮喘大鼠模型比较,呼吸道合胞病毒(RSV)诱导OVA致敏的哮喘模型大鼠喘息症状重,气道反应性明显升高,气道粘膜下炎症细胞浸润明显加重。呼吸道合胞病毒诱发哮喘模型大鼠BALF显示模型组、地塞米松和沙丁胺醇组、咳喘宁治疗组的BALF细胞总数及分类计数均升高,尤其以嗜酸性粒细胞升高为主,与PBS对照组比较,差异有显著性意义(P<0.01)咳喘宁治疗组、地沙治疗组细胞总数明显低于模型组,差异有显著性意义(P<0.01)；咳喘宁治疗组细胞总数稍高于地、沙治疗组,但差异无显著性意义(P>0.05)。与PBS对照组比较,模型组BALF中IL-4水平显著增高而IL-12水平显著降低(P<0.01)。咳喘宁治疗后IL-4水平与模型组比较显著下降(P<0.01),与PBS对照组之间比较差异无统计学意义(P>0.05)；IL-12水平与模型组比较显著增高(P<0.01)。地、沙组与咳喘宁治疗组IL-4、IL-12比较差异无统计学意义(P>0.05)。通过IL-4、IL-12与STAT6蛋白及其1mRNA的相关性分析,IL-4与STAT6蛋白和STAT6mRNA呈正相关(r值分别为0.941,0.703,P<0.01);IL-12与STAT6蛋白和STAT6mRNA呈负相关(r值分别为-0.906,-0.866,P<0.01)。免疫组化法和原位杂交法检测,哮喘模型大鼠支气管中STAT6蛋白和STAT6mRNA的表达均较PBS组增高(P<0.01),经咳喘宁治疗后,模型大鼠肺组织炎症程度明显减轻,支气管STAT6及其mRNA较模型组表达明显减弱(P<0.0J1)。呼吸道合胞病毒诱发哮喘模型大鼠CD4+T较PBS组增高明显(P<0.01)、CD8+T含量增高不显著(P>0.05),模型组CD4+T、CD8+T内STAT6的表达水平显著高于地沙组和咳喘宁治疗组(P<0.01),经咳喘宁治疗后CD4+T、CD8+T内STAT6的表达显著降低(P<0.01)。结论：咳喘宁能通过抑制病毒诱发哮喘模型大鼠支气管中STAT6及其mRNA的过度表达,下调CD4+T、CD8+T细胞内STAT6的表达,从而抑制BALF中IL-4的表达,纠正哮喘中Th2细胞的过度增殖分化,影响Th2免疫应答优势,并减少哮喘气道EOS的浸润,减轻模型大鼠气道炎症,降低其气道反应性,发挥防治病毒诱发哮喘的作用。更多还原

【Abstract】 This article demonstrated the mechanism of kechuanning decoction on the rat model of asthma induced by RSV with experimental techniques such as molecular biology、Organization immunology and virology. The results were as follows:Objective:To Study Kechuanning Decoction on Signal Transducer and Activator of Transcription6(STAT6) and its Gene Expression in the Rat Model of Asthma Induced by RSV.Methods:The asthmatic model was established by OVA-sensitization of intraperitoneal injection and repeated inhalation of OVA combination of several times of intranasal inoculation with RSV (1.010O6PFU/ml in50ul).Asthma Symptoms were observed. The change of airway responsiveness (represented by lung resistance RL) and function of lung (represented by PEF and the ratio of FEV0.4/FVC) were determined. Lung tissue sections were stained with Hematoxylin and eosin (H&E) for general morphology. Sixty young male SD rats were randomly divided into four groups:PBS control group with twenty rats; asthma model group with twenty rats; group treated with Dexamethasone and Salbutamol with ten rats and group treated with kechuanning decoction with ten rats respectively. In the experiment, the rat asthma model was established by the ovalbumin (OVA) challenge methods and stimulated with Respiratory syncytial virus.The protein expressions of STAT6were detected by immunohistochemistry techniques; the mRNA expressions of STAT6were detected by hybridization in situ. Cells in bronchoalvoelar lavage fluid (BALF) were counted and classified. And the supernatants of the BALF were used of detection of IL-4、IL-12. The correlation analysis of IL-4、IL-12and STAT6protein and its mRNA were compared. When the model was finished, isolated the PBMC from the peripheral blood, separated CD4+T、CD8+T lymphocyte with immunomagnetic beads. The purity and activity of CD4+T and CD8+T were measured by using flow cytometry trypan-blue dye exclusion test,etc.The protein expression of STAT6were detected by fluorescence-activated cell sorting.Results:Asthma symptoms were more severe in OVA/RSV group, compared with that of OVA group. RL increased and PEF、ratio of FEV0.4/FVC decreased in OVA/RSV group compared with those of OVA group (P<0.01).The bronchoconstriction and more inflammatory cells infiltration surrounding bronchi in the OVA/RSV group were found. Marked and extensive airway goblet cell hyperplasia and mucus retention in airway lumen in the OVA/RSV group were also observed. Immunohistochemistry and in situ hybridization showed that the expression of the protein content of STAT6and its mRNA around the bronchus of asthma group was significantly higher than that of the control PBS group and the treated group with kechuanning decoction and with Dexamethasone and Salbutamol (P<0.01), the epithelial cells were the chief expression cells; the expression of the protein content of STAT6and its mRNA of the treated group with kechuanning decoction was lower than the model asthma group (P<0.01) while a little higher than that of the treated group with Dexamethasone and Salbutamol, but it was no significance in statitics (P>0.05).Total BALF cells and more eosinophils in BALF from OVA/RSV group and the treated group with kechuanning decoction and with Dexamethasone and Salbutamol than those in PBS group were found (P<0.01). Total BALF cells were decreased in BALF from the treated group with kechuanning decoction and with Dexamethasone and Salbutamol than those in the asthma model group (P<0.01), but there were no differences in statistics between the treated group with kechuanning decoction and with Dexamethasone and Salbutamol. IL-4levels in BALF from the asthma model group increased greatly compared with the PBS group while IL-12levels were decreased (P<0.01), however IL-4levels in BALF from the treated group with kechuanning decoction decreased, compared with the PBS group, there were no significances in statistics (P>0.05)meanwhile the IL-12levels from the treated group with kechuanning decoction were increased compared with the asthma model group (P<0.01).There were positive correlation between IL-4、IL-12levels and the protein and the mRNA of STAT6(r=0.941,0.703respectively P<0.01),however negative correlation between IL-4、IL-12levels and the protein and the mRNA of STAT6(r=-0.906,-0.866respectively, P<0.01).The expression of the protein and the mRNA of STAT6were increased in the asthma model group, but it decreased in the treated group with kechuanning decoction. CD4+T lymphocyte in the asthma model group were increased however the hypertention of CD8+T lymphocyte were not significant (P>0.05),the expression of the protein of STAT6in the treated group with kechuanning decoction were decreased than it in the asthma model group (P<0.01)Conclusions:Kechuanning decoction could decrease the expression of the protein and the mRNA of STAT6in the bronchus and decrease the expression of the protein of STAT6in CD4+T、CD8+T lymphocyte, thereby it could regulate the disorder of Th1/Th2, it aimed to control the asthmatic attack.更多还原