本文選題：羅格列酮 + 肺纖維化��； 參考：《河北醫(yī)科大學(xué)》2013年碩士論文

【摘要】：研究背景及目的：特發(fā)性肺纖維化（idiopathic pulmonary fibrosis, IPF）是特發(fā)性間質(zhì)性肺炎中最常見(jiàn)的一種類型，肺纖維化目前發(fā)病機(jī)制尚不明確，病理以肺泡上皮細(xì)胞損傷，炎性細(xì)胞浸潤(rùn)，肌成纖維細(xì)胞聚集，細(xì)胞外基質(zhì)成分堆積為主要特征。因此，炎癥反應(yīng)與增生性改變被視為IPF的兩個(gè)重要階段。 羅格列酮屬于噻唑烷二酮類藥物，是過(guò)氧化物酶體增殖活化受體γ(PPAR-γ)的合成配體，，PPAR-γ被配體激活后進(jìn)而發(fā)揮生物學(xué)作用。臨床上PPAR-γ激動(dòng)劑多用于治療2型糖尿病，但近幾年研究表明PPARγ激動(dòng)劑羅格列酮能延緩肺纖維化進(jìn)程。羅格列酮可能通過(guò)調(diào)節(jié)炎癥反應(yīng)、調(diào)節(jié)巨噬細(xì)胞功能、促進(jìn)細(xì)胞凋亡及轉(zhuǎn)化、調(diào)節(jié)基底膜增厚反應(yīng)和細(xì)胞外基質(zhì)沉積等多種機(jī)制發(fā)揮對(duì)肺纖維化的抑制作用。博來(lái)霉素致大鼠肺纖維化模型證實(shí)了羅格列酮對(duì)肺纖維化的進(jìn)展有抑制作用。 纖溶酶原激活物抑制劑-1（Plasminogen activator inhibitor-1, PAI-1）是絲氨酸蛋白酶抑制劑(SERPIN)家族員之一,纖溶系統(tǒng)的主要抑制物,可迅速、有效地抑制尿激酶型纖溶酶原激活劑（uPA）和組織纖溶酶原激活劑（tPA）的活性，并且本身還能與細(xì)胞外基質(zhì)（ECM）的玻璃結(jié)合蛋白成分結(jié)合，使其成為ECM的一個(gè)組分，是調(diào)節(jié)纖溶系統(tǒng)促進(jìn)肺纖維化發(fā)生的中心環(huán)節(jié)。 目前國(guó)內(nèi)外已有關(guān)于PPAR-γ激動(dòng)劑羅格列酮可以抑制PAI-1的表達(dá)進(jìn)而延緩腎臟纖維化進(jìn)程的報(bào)道，本研究中初步探討羅格列酮是否通過(guò)抑制PAI-1的表達(dá)調(diào)節(jié)肺纖維化的病理生理過(guò)程，進(jìn)而延緩肺纖維化進(jìn)程，并初步探討羅格列酮延緩肺纖維化的可能信號(hào)機(jī)制。 方法： 1將本科室液氮凍存的大鼠胚肺成纖維細(xì)胞復(fù)蘇 2羅格列酮對(duì)成纖維細(xì)胞的影響及信號(hào)轉(zhuǎn)導(dǎo)通路 參照前人實(shí)驗(yàn)結(jié)果，羅格列酮最佳給藥濃度為30umol/L，根據(jù)我們前期研究結(jié)果，PAI-1最佳加藥濃度為20ug/ml，細(xì)胞分組：Control組、Rosiglitazone組(30umol/L)、 Rosiglitazone+PAI-1組（30umol/L+20ug/ml)，應(yīng)用western blotting法分別測(cè)定24h、48h和72h小時(shí)PAI-1、α-SMA、AKT、P-AKT、ERK、P-ERK蛋白的表達(dá)，RT-PCR分別測(cè)定24h的PAI-1、α-SMA、Ⅰ型膠原、Ⅲ型膠原mRNA的表達(dá)。 3統(tǒng)計(jì)學(xué)處理 數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差（X|-±SD）表示，采用社會(huì)科學(xué)統(tǒng)計(jì)程序（StatisticalProgrom for Social Sciences13.0）進(jìn)行統(tǒng)計(jì)學(xué)分析，觀察組與對(duì)照組的樣本均數(shù)比較采用單因素方差分析，P0.05為差異有統(tǒng)計(jì)學(xué)意義。比較組間差異，有顯著差異者用S-N-K法進(jìn)行兩兩比較，P0.05為差異有統(tǒng)計(jì)學(xué)意義。 結(jié)果： 1成纖維細(xì)胞經(jīng)復(fù)蘇呈現(xiàn)圓球形約在4-5小時(shí)后貼壁，開始延伸，完全伸展后的細(xì)胞為梭形，細(xì)胞透亮，緊密連接，呈放射狀生長(zhǎng)，相互連接，經(jīng)復(fù)蘇后的細(xì)胞是第2代，實(shí)驗(yàn)選用3-5代。 2經(jīng)過(guò)羅格列酮干預(yù)后Rosiglitazone組肺成纖維細(xì)胞PAI-1的蛋白表達(dá)在24h較Control組即明顯降低，且作用可持續(xù)至72h；并且Rosiglitazone組肺成纖維細(xì)胞PAI-1的mRNA表達(dá)同樣較Control組明顯降低（P0.05）。 3Rosiglitazone組肺成纖維細(xì)胞α-SMA的mRNA及蛋白含量較Control組均明顯降低；增加外源性PAI-1后，Rosiglitazone+PAI-1組的α-SMA的mRNA及蛋白含量較Rosiglitazone組明顯增加（P0.05）。 4Rosiglitazone組肺成纖維細(xì)胞Ⅰ型、Ⅲ型膠原的mRNA含量較Control組均明顯降低；增加外源性PAI-1后Rosiglitazone+PAI-1組的Ⅰ型、Ⅲ型膠原的mRNA含量較Rosiglitazone組明顯增加（P0.05）。 5Control組、Rosiglitazone組、Rosiglitazone+PAI-1組的AKT、P-AKT的含量無(wú)明顯改變（P0.05）；Rosiglitazone組肺成纖維細(xì)胞ERK、P-ERK的蛋白含量較Control組均明顯下調(diào)（P0.05）。 結(jié)論： 1羅格列酮能抑制大鼠胚肺成纖維細(xì)胞向肌成纖維細(xì)胞的轉(zhuǎn)化及膠原的合成。 2羅格列酮可以抑制大鼠胚肺成纖維細(xì)胞PAI-1的表達(dá)，從而使纖溶活性增加。 3增加外源性PAI-1降低纖溶系統(tǒng)活性后，羅格列酮抑制大鼠胚肺成纖維細(xì)胞向肌成纖維細(xì)胞的轉(zhuǎn)化及膠原的合成作用減弱。 4羅格列酮對(duì)大鼠胚肺成纖維細(xì)胞的調(diào)節(jié)可能是通過(guò)ERK/P-ERK信號(hào)通路實(shí)現(xiàn)的。
[Abstract]:Background and purpose: idiopathic pulmonary fibrosis (IPF) is the most common type of idiopathic interstitial pneumonia. The pathogenesis of pulmonary fibrosis is not yet clear. The pathology of pulmonary fibrosis is alveolar epithelial cell damage, inflammatory cell infiltration, myofibroblast aggregation, and accumulation of extracellular matrix components as the main characteristics. Therefore, inflammatory reaction and proliferative change are regarded as the two important stages of IPF.
Rosiglitazone is a thiazolidane two ketone, a synthetic ligand for the peroxisome proliferator activated receptor gamma (PPAR- gamma), and PPAR- gamma is activated by the ligand and plays a biological role. Clinical PPAR- gamma agonists are used for the treatment of type 2 diabetes, but in recent years, the study showed that the PPAR gamma agonist rosiglitazone could delay the process of pulmonary fibrosis. Glipone may regulate the function of macrophages, regulate the function of macrophages, promote cell apoptosis and transformation, regulate the thickening reaction of the basement membrane and the deposition of extracellular matrix to inhibit the pulmonary fibrosis. The rat pulmonary fibrosis model induced by bleomycin has confirmed that roglitone has inhibitory effect on the progression of pulmonary fibrosis.
The plasminogen activator inhibitor -1 (Plasminogen activator inhibitor-1, PAI-1) is one of the serine protease inhibitor (SERPIN) family members, the main inhibitor of the fibrinolytic system, which can quickly and effectively inhibit the activity of the urokinase type plasminogen activator (uPA) and tissue plasminogen activator (tPA), and it itself can also be used outside the cell. The binding of matrix bound (ECM) glass binding protein makes it a component of ECM. It is a central link in regulating fibrinolytic system to promote pulmonary fibrosis.
At present, there have been reports that PPAR- gamma agonist rosiglitazone can inhibit the expression of PAI-1 and then delay the process of renal fibrosis. In this study, it is preliminarily discussed whether rosiglitazone can regulate the pathophysiological process of pulmonary fibrosis by inhibiting the expression of PAI-1, and then delaying the process of pulmonary fibrosis and preliminary study of rosiglitazone to delay the lung. The possible signaling mechanism of fibrosis.
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