【摘要】：目的：探討鈣蛋白酶（calpain）以及其下游的PI3K/Akt信號通路在氣道炎癥導致的哮喘氣道平滑肌重建中的作用。 方法：以卵清蛋白被動致敏的方法復制小鼠慢性支氣管哮喘氣道重構的模型，并腹腔注射calpain抑制劑calpeptin，檢測肺組織calpain活性，并用Masson染色檢測支氣管膠原含量以及平滑肌厚度。用哮喘相關細胞因子IL-4，IL-5和TNF-α處理原代培養(yǎng)支氣管平滑肌細胞，檢測calpain活性， Western blot方法檢測Ⅰ型膠原和磷酸化的Akt（p-Akt）的蛋白表達，以及BrdU-ELISA方法檢測平滑肌細胞增殖。同時用calpain抑制劑MDL28170、PI3K抑制劑LY294002分別阻斷calpain、Akt信號，進一步證實該通路在膠原生成和細胞增殖中的作用。 結果：哮喘小鼠肺組織calpain活性明顯高于對照組，Masson染色結果顯示支氣管有明顯的膠原沉積以及平滑肌增厚，而使用calpain抑制劑的哮喘+calpeptin組小鼠肺組織calpain活性低于哮喘組，，而且膠原含量和平滑肌層厚度均低于哮喘組。哮喘細胞因子IL-4，IL-5及TNF-α處理，均可促使氣道平滑肌細胞calpain活性增加、細胞增殖以及Ⅰ型膠原和p-Akt表達。 Calpain抑制劑MDL28170抑制Akt的磷酸化（p-Akt），而且，MDL28170及PI3K抑制劑LY294002均可減輕哮喘細胞因子引起的細胞增殖和Ⅰ型膠原表達。 結論： Calpain在支氣管哮喘氣道平滑肌細胞重建中起重要作用，calpain活化PI3K/Akt信號通路是其機制之一。
[Abstract]:Aim: to investigate the role of calpain (calpain) and its downstream PI3K/Akt signaling pathway in airway smooth muscle remodeling induced by airway inflammation. Methods: the airway remodeling model of chronic bronchial asthma in mice was induced by passive sensitization of ovalbumin. The lung tissue calpain activity was detected by intraperitoneal injection of calpain inhibitor calpeptin, and the content of bronchial collagen and the thickness of smooth muscle were detected by Masson staining. Bronchial smooth muscle cells were treated with Asthma related cytokines (IL-4,IL-5 and TNF- 偽). The expression of type I collagen and phosphorylated Akt (p-Akt) protein was detected by calpain activity, Western blot method, and the proliferation of smooth muscle cells was detected by BrdU-ELISA method. At the same time, the calpain,Akt signal was blocked by LY294002, a calpain inhibitor, MDL28170,PI3K inhibitor, to further confirm the role of this pathway in collagen production and cell proliferation. Results: the calpain activity of lung tissue in asthmatic mice was significantly higher than that in control group. The results showed that there was obvious collagen deposition and smooth muscle thickening in the bronchus, while the calpain activity in lung tissue of asthmatic calpeptin group with calpain inhibitor was lower than that of asthmatic calpeptin group. And collagen content and smooth muscle layer thickness were lower than asthma group. The treatment of asthma cytokines IL-4,IL-5 and TNF- 偽 could increase the calpain activity, cell proliferation and the expression of type I collagen and p-Akt in airway smooth muscle cells. MDL28170, an inhibitor of Calpain, inhibited the phosphorylation of Akt (p-Akt), and both MDL28170 and LY294002, the inhibitor of PI3K, could attenuate the proliferation and type I collagen expression induced by cytokines of asthma. Conclusion: Calpain plays an important role in the remodeling of airway smooth muscle cells in bronchial asthma. Calpain activates PI3K/Akt signaling pathway is one of its mechanisms.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R562.25

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