基于IL-4、IL-13膜受体表达调控STAT-6信号通路探讨平哮颗粒治疗支气管哮喘大鼠模型的作用机理研究

发布时间：2018-01-12 07:26

  本文关键词：基于IL-4、IL-13膜受体表达调控STAT-6信号通路探讨平哮颗粒治疗支气管哮喘大鼠模型的作用机理研究　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

  更多相关文章： 平哮颗粒 哮喘 STAT-6 IL-4 L-4Rα IL-13Rα1/α2

【摘要】：背景:平哮颗粒是导师史锁芳教授治疗支气管哮喘的经验方,前期临床观察显示其疗效确切,但其作用机制目前尚不明确,本研究立足于IL-4Rα、IL-13Rα 1、IL-13Rα 2的表达进而调控STAT-6的信号通路,探讨平哮颗粒治疗支气管哮喘的作用机理。目的:阐明平哮颗粒通过IL-4、IL-13膜受体表达调控STAT-6信号通路治疗支气管哮喘的作用机理,同时为临床运用平哮颗粒有效治疗哮喘提供分子生物学佐证。方法:清洁级SD大鼠60只,随机平均分为空白组、哮喘模型组、地塞米松组、平哮低剂量组、平哮高剂量组等五组,每组雌雄各半。除空白组以外,各组腹腔注射"鸡卵蛋白+Al(OH)_3混悬液"和灭活百日咳杆菌致敏,并通过OVA雾化吸入诱发哮喘,建立模型;空白组予腹腔注射等量生理盐水,并且予蒸馏水雾化吸入作为对照组。模型建立后,实验各组分别给予对应的药物治疗,空白组给予蒸馏水灌胃以作对照。实验过程中密切观察大鼠症状,并通过哮喘行为学评分评估。药物干预14天后,处死大鼠,取大鼠血清及肺组织标本。检测:1)肺组织H.E染色,光镜下观察病理变化;2)Giemsa染色,光镜下观察嗜酸性粒细胞浸润情况;3)ELISA法检测血清IL-4、IL-12、IL-13浓度变化;4)Westerm-Blot检测肺组织pSTAT-6表达情况;RT-PCR检测肺组织IL-4R α、IL-13 R α 1/α 2表达情况。结果:1)实验采用OVA、Al(OH)_3和百日咳杆菌等联合致敏、诱喘的造模法,切合哮喘动物模型基本特征,符合实验研究要求,构建过敏性哮喘大鼠模型成功。2)HE染色:空白组肺组织未见明显改变;模型组支气管壁明显增厚,管腔内现渗出物,肺间质现嗜酸性粒细胞、中性粒细胞等大量炎细胞浸润,纤维结缔组织增生等;其余三组可见少数支气管平滑肌轻度增厚,肺间质现少许炎细胞浸润。3)Giemsa染色:模型组可见较多嗜酸性粒细胞浸润,空白组未见明显异常,其余各可见少许嗜酸性粒细胞浸润。4)ELISA检测:①IL-4水平比较:五组之间并未见明显差异,无显著统计学意义(P0.05)。②IL-13水平比较:与模型组相比,地米组IL-13水平显著偏低(P0.05),但模型组与其余各组IL-13水平相当,空白组与其他组水平相当,无统计学意义。③IL-12测出OD值几乎都为负值,无意义。5)Western Blot检测:哮喘大鼠STAT-6水平五组分析比较显示,模型组与中高、地米、空白组三组存在差异(p0.05),并且模型组与空白组存在显著性差异(p0.01),但模型组与中低组水平相当(P0.05);空白组STAT-6水平与其余四组存在显著性差异(p0.01)。中高组与地米组无明显差异,中药高低两组水平相当(P0.05)。6)RT-PCR检测:①L-4RmRNA比较:模型组明显高于其余四组(P0.05),且模型组和地米组、中药组以及空白组存在显著性差异(p0.01);而空白组明显低于模型和中低组,且差异显著(P0.01),但与其余两组水平相当,无统计学意义;中高组与地米组和中低组无明显差异。②IL-13Rα1 mRNA比较:模型组明显高于其余四组,差异有统计学意义(p0.05),且空白组、地米组及中高组与模型组比较有显著性差异(p0.01);空白组明显低于模型、中高、中低组,差异有统计学意义(p0.05),并且模型、中低组先与空白组有显著性差异(p0.01);地米组与中高组水平相当,无明显差异(P0.05),但与中低组比存在差异性,有统计学意义(p0.05);中药高低两组水平相当,无统计学意义(P0.05)。③IL-13Rα2mRNA比较:模型组水平明显高于其余四组,具有显著统计学意义(P0.05),并且模型组与地米组、中高组及空白组存在显著性差异(p0.01);空白组与模型组比较,水平明显偏低,且差异显著(P0.01),但与其余三组水平相当,无明显差异(P0.05)。地米、中高、中低三组水平相当(p0.05)。结论:大鼠肺组织病理和相关炎症指标经平哮颗粒和地塞米松干预治疗后得到改善。平哮颗粒通过分别抑制IL-4和IL-13的膜受体IL-4R、IL-13 Rα 1/α 2,从而抑制STAT-6磷酸化,减少Th2型细胞因子释放水平,使炎症反应向Th1方向偏移。这可能是平哮颗粒治疗过敏性哮喘的机制之一。
[Abstract]:Background: Pingxiao granule is the experience of Professor Shi Suofang in the treatment of bronchial asthma, the curative effect observation of early clinical, but its mechanism is not clear. This research is based on the IL-4R IL-13R alpha, alpha 1, alpha 2 expression of IL-13R signaling pathway and the regulation of STAT-6, to explore the mechanism of Pingxiao granule in treating bronchial asthma. Objective: to clarify the Pingxiao granule by IL-4, expression of IL-13 membrane receptor mechanism of STAT-6 signaling pathway in the treatment of bronchial asthma, and to provide evidence for the clinical application of molecular biology of Pingxiao granule is effective in the treatment of asthma. Methods: 60 clean grade SD rats were randomly divided into control group, asthmatic model group, dexamethasone Pingxiao group, low dose group, high dose group five Pingxiao group, each group of male and female. In addition to the blank group, the intraperitoneal injection of ovalbumin (+Al OH) _3 suspension and inactivation caused by Bordetella pertussis Min, and through inhalation of OVA induced asthma model was established; the control group were treated with intraperitoneal injection of normal saline and distilled water inhalation as the control group. After the establishment of the model, experimental groups were given corresponding drug treatment, the control group was given distilled water as control group. During the experiment, close observation of symptoms of rats and, through the asthma behavior score assessment. 14 days after drug intervention, rats were killed, serum and lung tissue of rats were detected: 1). Lung tissue H.E staining, pathological changes were observed under light microscope; 2) Giemsa staining, light microscope to observe the infiltration of eosinophils; 3) serum IL-4, ELISA IL-12, IL-13 concentration; 4) Westerm-Blot detection of lung tissue pSTAT-6 expression; RT-PCR detection of lung tissue IL-4R IL-13 R alpha, alpha 1/ alpha 2 expression. Results: 1) using OVA, Al (OH) _3 and whooping cough bacili combined sensitization, theprovocation made Model method, basic characteristics with the animal model of asthma, consistent with experimental study, constructing allergic asthma rat model successfully.2) HE staining: the lung tissue of control group had no obvious change; model group, bronchial wall thickening, the lumen of the exudate interstitial lung are eosinophils, neutrophils and other inflammatory cell infiltration, fibrous connective tissue hyperplasia; the other three groups showing a few slight thickening of bronchial smooth muscle, lung interstitial infiltration of inflammatory cells is a little.3) Giemsa staining: the model group showed more eosinophil infiltration, the blank group had no obvious abnormalities, the rest of the visible little eosinophil infiltration).4 ELISA detection: a comparison the level of IL-4 between the five groups and no significant difference was not significant (P0.05). The IL-13 level: compared with the model group, dexamethasone group IL-13 levels were significantly lower (P0.05), but the model group and other groups IL-13 Rather, a blank group and other groups, no statistical significance. The IL-12 measured od almost all negative, no significance of.5 Blot detection: Western) comparison analysis of five groups of STAT-6 levels in asthmatic rats, the model group and the high, to three meters, the blank group group differences (P0.05), and model group and control group had significant difference (P0.01), but the model group and low level group (P0.05); STAT-6 control group and the other four groups had significant difference (P0.01). No significant difference in the high group and dexamethasone group, Chinese medicine group of two water level is equal to (P0.05).6) RT-PCR detection: L-4RmRNA: model group was significantly higher than the other four groups (P0.05), and model group and dexamethasone group, there was significant difference between the Chinese medicine group and blank group (P0.01); and the blank group was significantly lower than the model group and the low, and the difference was significant (P0.01), but with the other two groups the same level, no statistical significance 涔，

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