罗格列酮抑制大鼠胚肺成纤维细胞转化及胶原合成的机制

发布时间：2018-06-07 19:00

  本文选题：罗格列酮 + 肺纤维化　； 参考：《河北医科大学》2013年硕士论文

【摘要】：研究背景及目的：特发性肺纤维化（idiopathic pulmonary fibrosis, IPF）是特发性间质性肺炎中最常见的一种类型，肺纤维化目前发病机制尚不明确，病理以肺泡上皮细胞损伤，炎性细胞浸润，肌成纤维细胞聚集，细胞外基质成分堆积为主要特征。因此，炎症反应与增生性改变被视为IPF的两个重要阶段。 罗格列酮属于噻唑烷二酮类药物，是过氧化物酶体增殖活化受体γ(PPAR-γ)的合成配体，，PPAR-γ被配体激活后进而发挥生物学作用。临床上PPAR-γ激动剂多用于治疗2型糖尿病，但近几年研究表明PPARγ激动剂罗格列酮能延缓肺纤维化进程。罗格列酮可能通过调节炎症反应、调节巨噬细胞功能、促进细胞凋亡及转化、调节基底膜增厚反应和细胞外基质沉积等多种机制发挥对肺纤维化的抑制作用。博来霉素致大鼠肺纤维化模型证实了罗格列酮对肺纤维化的进展有抑制作用。 纤溶酶原激活物抑制剂-1（Plasminogen activator inhibitor-1, PAI-1）是丝氨酸蛋白酶抑制剂(SERPIN)家族员之一,纤溶系统的主要抑制物,可迅速、有效地抑制尿激酶型纤溶酶原激活剂（uPA）和组织纤溶酶原激活剂（tPA）的活性，并且本身还能与细胞外基质（ECM）的玻璃结合蛋白成分结合，使其成为ECM的一个组分，是调节纤溶系统促进肺纤维化发生的中心环节。 目前国内外已有关于PPAR-γ激动剂罗格列酮可以抑制PAI-1的表达进而延缓肾脏纤维化进程的报道，本研究中初步探讨罗格列酮是否通过抑制PAI-1的表达调节肺纤维化的病理生理过程，进而延缓肺纤维化进程，并初步探讨罗格列酮延缓肺纤维化的可能信号机制。 方法： 1将本科室液氮冻存的大鼠胚肺成纤维细胞复苏 2罗格列酮对成纤维细胞的影响及信号转导通路 参照前人实验结果，罗格列酮最佳给药浓度为30umol/L，根据我们前期研究结果，PAI-1最佳加药浓度为20ug/ml，细胞分组：Control组、Rosiglitazone组(30umol/L)、 Rosiglitazone+PAI-1组（30umol/L+20ug/ml)，应用western blotting法分别测定24h、48h和72h小时PAI-1、α-SMA、AKT、P-AKT、ERK、P-ERK蛋白的表达，RT-PCR分别测定24h的PAI-1、α-SMA、Ⅰ型胶原、Ⅲ型胶原mRNA的表达。 3统计学处理 数据用均数±标准差（X|-±SD）表示，采用社会科学统计程序（StatisticalProgrom for Social Sciences13.0）进行统计学分析，观察组与对照组的样本均数比较采用单因素方差分析，P0.05为差异有统计学意义。比较组间差异，有显著差异者用S-N-K法进行两两比较，P0.05为差异有统计学意义。 结果： 1成纤维细胞经复苏呈现圆球形约在4-5小时后贴壁，开始延伸，完全伸展后的细胞为梭形，细胞透亮，紧密连接，呈放射状生长，相互连接，经复苏后的细胞是第2代，实验选用3-5代。 2经过罗格列酮干预后Rosiglitazone组肺成纤维细胞PAI-1的蛋白表达在24h较Control组即明显降低，且作用可持续至72h；并且Rosiglitazone组肺成纤维细胞PAI-1的mRNA表达同样较Control组明显降低（P0.05）。 3Rosiglitazone组肺成纤维细胞α-SMA的mRNA及蛋白含量较Control组均明显降低；增加外源性PAI-1后，Rosiglitazone+PAI-1组的α-SMA的mRNA及蛋白含量较Rosiglitazone组明显增加（P0.05）。 4Rosiglitazone组肺成纤维细胞Ⅰ型、Ⅲ型胶原的mRNA含量较Control组均明显降低；增加外源性PAI-1后Rosiglitazone+PAI-1组的Ⅰ型、Ⅲ型胶原的mRNA含量较Rosiglitazone组明显增加（P0.05）。 5Control组、Rosiglitazone组、Rosiglitazone+PAI-1组的AKT、P-AKT的含量无明显改变（P0.05）；Rosiglitazone组肺成纤维细胞ERK、P-ERK的蛋白含量较Control组均明显下调（P0.05）。 结论： 1罗格列酮能抑制大鼠胚肺成纤维细胞向肌成纤维细胞的转化及胶原的合成。 2罗格列酮可以抑制大鼠胚肺成纤维细胞PAI-1的表达，从而使纤溶活性增加。 3增加外源性PAI-1降低纤溶系统活性后，罗格列酮抑制大鼠胚肺成纤维细胞向肌成纤维细胞的转化及胶原的合成作用减弱。 4罗格列酮对大鼠胚肺成纤维细胞的调节可能是通过ERK/P-ERK信号通路实现的。
[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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