發(fā)布時間：2018-01-12 07:26

  本文關(guān)鍵詞：基于IL-4、IL-13膜受體表達(dá)調(diào)控STAT-6信號通路探討平哮顆粒治療支氣管哮喘大鼠模型的作用機(jī)理研究　出處：《南京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 平哮顆粒 哮喘 STAT-6 IL-4 L-4Rα IL-13Rα1/α2

【摘要】：背景:平哮顆粒是導(dǎo)師史鎖芳教授治療支氣管哮喘的經(jīng)驗(yàn)方,前期臨床觀察顯示其療效確切,但其作用機(jī)制目前尚不明確,本研究立足于IL-4Rα、IL-13Rα 1、IL-13Rα 2的表達(dá)進(jìn)而調(diào)控STAT-6的信號通路,探討平哮顆粒治療支氣管哮喘的作用機(jī)理。目的:闡明平哮顆粒通過IL-4、IL-13膜受體表達(dá)調(diào)控STAT-6信號通路治療支氣管哮喘的作用機(jī)理,同時為臨床運(yùn)用平哮顆粒有效治療哮喘提供分子生物學(xué)佐證。方法:清潔級SD大鼠60只,隨機(jī)平均分為空白組、哮喘模型組、地塞米松組、平哮低劑量組、平哮高劑量組等五組,每組雌雄各半。除空白組以外,各組腹腔注射"雞卵蛋白+Al(OH)_3混懸液"和滅活百日咳桿菌致敏,并通過OVA霧化吸入誘發(fā)哮喘,建立模型;空白組予腹腔注射等量生理鹽水,并且予蒸餾水霧化吸入作為對照組。模型建立后,實(shí)驗(yàn)各組分別給予對應(yīng)的藥物治療,空白組給予蒸餾水灌胃以作對照。實(shí)驗(yàn)過程中密切觀察大鼠癥狀,并通過哮喘行為學(xué)評分評估。藥物干預(yù)14天后,處死大鼠,取大鼠血清及肺組織標(biāo)本。檢測:1)肺組織H.E染色,光鏡下觀察病理變化;2)Giemsa染色,光鏡下觀察嗜酸性粒細(xì)胞浸潤情況;3)ELISA法檢測血清IL-4、IL-12、IL-13濃度變化;4)Westerm-Blot檢測肺組織pSTAT-6表達(dá)情況;RT-PCR檢測肺組織IL-4R α、IL-13 R α 1/α 2表達(dá)情況。結(jié)果:1)實(shí)驗(yàn)采用OVA、Al(OH)_3和百日咳桿菌等聯(lián)合致敏、誘喘的造模法,切合哮喘動物模型基本特征,符合實(shí)驗(yàn)研究要求,構(gòu)建過敏性哮喘大鼠模型成功。2)HE染色:空白組肺組織未見明顯改變;模型組支氣管壁明顯增厚,管腔內(nèi)現(xiàn)滲出物,肺間質(zhì)現(xiàn)嗜酸性粒細(xì)胞、中性粒細(xì)胞等大量炎細(xì)胞浸潤,纖維結(jié)締組織增生等;其余三組可見少數(shù)支氣管平滑肌輕度增厚,肺間質(zhì)現(xiàn)少許炎細(xì)胞浸潤。3)Giemsa染色:模型組可見較多嗜酸性粒細(xì)胞浸潤,空白組未見明顯異常,其余各可見少許嗜酸性粒細(xì)胞浸潤。4)ELISA檢測:①IL-4水平比較:五組之間并未見明顯差異,無顯著統(tǒng)計學(xué)意義(P0.05)。②IL-13水平比較:與模型組相比,地米組IL-13水平顯著偏低(P0.05),但模型組與其余各組IL-13水平相當(dāng),空白組與其他組水平相當(dāng),無統(tǒng)計學(xué)意義。③IL-12測出OD值幾乎都為負(fù)值,無意義。5)Western Blot檢測:哮喘大鼠STAT-6水平五組分析比較顯示,模型組與中高、地米、空白組三組存在差異(p0.05),并且模型組與空白組存在顯著性差異(p0.01),但模型組與中低組水平相當(dāng)(P0.05);空白組STAT-6水平與其余四組存在顯著性差異(p0.01)。中高組與地米組無明顯差異,中藥高低兩組水平相當(dāng)(P0.05)。6)RT-PCR檢測:①L-4RmRNA比較:模型組明顯高于其余四組(P0.05),且模型組和地米組、中藥組以及空白組存在顯著性差異(p0.01);而空白組明顯低于模型和中低組,且差異顯著(P0.01),但與其余兩組水平相當(dāng),無統(tǒng)計學(xué)意義;中高組與地米組和中低組無明顯差異。②IL-13Rα1 mRNA比較:模型組明顯高于其余四組,差異有統(tǒng)計學(xué)意義(p0.05),且空白組、地米組及中高組與模型組比較有顯著性差異(p0.01);空白組明顯低于模型、中高、中低組,差異有統(tǒng)計學(xué)意義(p0.05),并且模型、中低組先與空白組有顯著性差異(p0.01);地米組與中高組水平相當(dāng),無明顯差異(P0.05),但與中低組比存在差異性,有統(tǒng)計學(xué)意義(p0.05);中藥高低兩組水平相當(dāng),無統(tǒng)計學(xué)意義(P0.05)。③IL-13Rα2mRNA比較:模型組水平明顯高于其余四組,具有顯著統(tǒng)計學(xué)意義(P0.05),并且模型組與地米組、中高組及空白組存在顯著性差異(p0.01);空白組與模型組比較,水平明顯偏低,且差異顯著(P0.01),但與其余三組水平相當(dāng),無明顯差異(P0.05)。地米、中高、中低三組水平相當(dāng)(p0.05)。結(jié)論:大鼠肺組織病理和相關(guān)炎癥指標(biāo)經(jīng)平哮顆粒和地塞米松干預(yù)治療后得到改善。平哮顆粒通過分別抑制IL-4和IL-13的膜受體IL-4R、IL-13 Rα 1/α 2,從而抑制STAT-6磷酸化,減少Th2型細(xì)胞因子釋放水平,使炎癥反應(yīng)向Th1方向偏移。這可能是平哮顆粒治療過敏性哮喘的機(jī)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，

本文編號：1413279