發(fā)布時(shí)間：2018-10-26 18:23

【摘要】：近年來，，作為哮喘慢性化、嚴(yán)重化、持續(xù)化的病理基礎(chǔ)，氣道重塑在哮喘防治中的潛在作用被廣為關(guān)注。它不僅是激素抵抗性哮喘的病理基礎(chǔ)，也是不可逆性氣道阻塞和氣道高反應(yīng)性的病理基礎(chǔ)。作為Vit-D在體內(nèi)極其重要的的代謝產(chǎn)物之一，骨化三醇（羅蓋全，Rocalirol）在哮喘的治療中不僅對(duì)于氣道慢性炎癥而且對(duì)于免疫應(yīng)答均起到一定的調(diào)節(jié)作用。但至今國內(nèi)外均未見關(guān)于骨化三醇對(duì)哮喘氣道重塑的報(bào)道。該研究設(shè)計(jì)從體內(nèi)、外兩個(gè)方面觀察骨化三醇對(duì)支氣管哮喘氣道重塑的影響，并進(jìn)一步探討其作用機(jī)制，為推廣其臨床應(yīng)用提供依據(jù)。 該項(xiàng)內(nèi)容分為兩個(gè)部分： 第一部分骨化三醇調(diào)節(jié)哮喘小鼠氣道重塑及NF-κB信號(hào)通路的研究 目的：觀察骨化三醇對(duì)哮喘小鼠氣道重塑的影響，并進(jìn)一步探討其機(jī)制。方法：實(shí)驗(yàn)小鼠隨機(jī)分為對(duì)照組、哮喘組和VD組。采用卵白蛋白致敏及激發(fā)的方法制備小鼠哮喘氣道重塑的模型；選擇HE染色法觀察氣道壁病理改變；Masson染色觀察氣道壁膠原染色情況，AB-PAS染色觀察杯狀細(xì)胞增生情況，計(jì)算機(jī)圖像分析系統(tǒng)測(cè)定氣道形態(tài)學(xué)指標(biāo)，蛋白免疫印記法檢測(cè)NF-κB p65的蛋白水平。結(jié)果：(1)骨化三醇對(duì)哮喘氣道重塑具有部分逆轉(zhuǎn)的作用；(2)骨化三醇能明顯抑制哮喘肺組織NF-κB p65的核易位。結(jié)論：骨化三醇的干預(yù)可顯著減輕哮喘氣道重塑的病理改變；這一作用可能與其對(duì)哮喘肺內(nèi)NF-κB活化的抑制作用有關(guān)。 第二部分骨化三醇對(duì)哮喘小鼠氣道平滑肌細(xì)胞凋亡及NF-κB信號(hào)通路的影響 目的：檢測(cè)骨化三醇對(duì)哮喘小鼠氣道平滑肌細(xì)胞（airway smooth musclecells,ASMCs）的凋亡及其核轉(zhuǎn)錄因子-κB信號(hào)通路的影響，從細(xì)胞及分子兩個(gè)水平探究Rocalirol對(duì)哮喘氣道重塑的可能作用機(jī)制。方法：原代培養(yǎng)正常及哮喘小鼠ASMCs，以Rocalirol作為干預(yù)因素。流式細(xì)胞儀測(cè)定細(xì)胞凋亡，熒光顯微鏡觀察細(xì)胞凋亡形態(tài)學(xué)變化；凝膠遷移滯后實(shí)驗(yàn)(EMSA法)檢測(cè)NF-κB的DNA結(jié)合活性；實(shí)時(shí)熒光定量RT-PCR法和蛋白免疫印記法檢測(cè)IκBα的表達(dá)。結(jié)果：(1)10 7M Rocalirol可顯著誘導(dǎo)哮喘小鼠ASMCs的凋亡；(2) Rocalirol可顯著削弱哮喘小鼠ASMCs中NF-κB的DNA結(jié)合活性；(3)Rocalirol可增加哮喘小鼠ASMCs中IκBα的蛋白及mRNA表達(dá)。結(jié)論：Rocalirol可直接誘導(dǎo)哮喘ASMCs的凋亡，這種作用可能與NF-κB信號(hào)通路受骨化三醇抑制有關(guān)。
[Abstract]:In recent years, as the pathological basis of chronic, severe and persistent asthma, the potential role of airway remodeling in the prevention and treatment of asthma has been widely concerned. It is not only the pathological basis of hormone resistant asthma, but also the pathological basis of irreversible airway obstruction and airway hyperresponsiveness. As one of the most important metabolites of Vit-D in vivo, roguetriol (, Rocalirol) plays an important role not only in chronic airway inflammation but also in immune response in the treatment of asthma. However, there is no report on airway remodeling of asthma by ossifying triol at home and abroad. The purpose of this study was to investigate the effect of ossifying triol on airway remodeling of bronchial asthma in vivo and in vitro, and to explore its mechanism. The content is divided into two parts: the first part is the effect of ossifying triol on airway remodeling and NF- 魏 B signaling pathway in asthmatic mice objective: to observe the effect of ossifying triol on airway remodeling in asthmatic mice. The mechanism is further discussed. Methods: the mice were randomly divided into control group, asthma group and VD group. The airway remodeling model of mouse asthma was established by sensitizing and stimulating ovalbumin, and the pathological changes of airway wall were observed by HE staining. Collagen staining in airway wall was observed by Masson staining, goblet cell proliferation was observed by AB-PAS staining, airway morphology was measured by computer image analysis system, and the protein level of NF- 魏 B p65 was detected by protein imprinting. Results: (1) ossifying triol could partially reverse airway remodeling of asthma, (2) ossification triol could significantly inhibit the nuclear translocation of NF- 魏 B p65 in lung tissue of asthma. Conclusion: the intervention of calcitriol can significantly attenuate the pathological changes of airway remodeling in asthma, which may be related to its inhibitory effect on the activation of NF- 魏 B in the lung of asthma. Part two effects of ossifying triol on apoptosis of airway smooth muscle cells and NF- 魏 B signaling pathway in asthmatic mice objective: to detect the effect of oscification triol on (airway smooth musclecells, of airway smooth muscle cells of asthmatic mice ASMCs) apoptosis and the effect of nuclear transcription factor-魏 B signaling pathway. To explore the possible mechanism of Rocalirol on airway remodeling in asthmatic patients at both cellular and molecular levels. Methods: Rocalirol was used as an intervention factor in primary cultured normal and asthmatic mice ASMCs,. Apoptosis was measured by flow cytometry, morphological changes of apoptosis were observed by fluorescence microscope, DNA binding activity of NF- 魏 B was detected by gel migration lag assay (EMSA). The expression of I 魏 B 偽 was detected by real time fluorescence quantitative RT-PCR and protein imprinting. Results: (1) 10 渭 m Rocalirol significantly induced the apoptosis of ASMCs in asthmatic mice; (2) Rocalirol significantly reduced the DNA binding activity of NF- 魏 B in ASMCs of asthmatic mice; (3) Rocalirol increased the expression of I 魏 B 偽 and mRNA in ASMCs of asthmatic mice. Conclusion: Rocalirol can directly induce the apoptosis of ASMCs in asthma. This effect may be related to the inhibition of NF- 魏 B signaling pathway by oscitic triol.
【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R562.25

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