纤溶酶原激活物抑制剂-1对大鼠胚肺成纤维细胞增殖和凋亡的影响及其机制

发布时间：2018-04-23 05:40

  本文选题：肺纤维化 + 成纤维细胞　； 参考：《河北医科大学》2012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，约占47%～71%，其预后差，生存中位时间仅为3至4年。IPF主要病理特征为肺泡Ⅰ型上皮细胞、血管内皮细胞及基底膜的损害，并伴有嗜中性粒细胞、巨噬细胞和淋巴细胞等炎症细胞浸润，同时肺泡Ⅱ型上皮细胞与成纤维细胞异常增生，胶原过度沉积。IPF的基本病因目前还不清楚，在以往的研究中多认为与多种细胞因子参与的炎症和纤维化过程有关，如:转化生长因子-β(TGF-β)、肿瘤坏死因子-α(TNF-α)、血小板源性生长因子(PDGF)、基质金属蛋白酶MMP等。 纤溶酶原激活物抑制剂-1（Plasminogen activator inhibitor-1, PAI-1）的主要作用是抑制尿激酶型纤溶酶原激活物(urokinase-type plasminogenactivator, uPA)和组织纤维蛋白溶酶原激活剂(tissue-type plasminogenactivators, tPA)，它与u-PA或t-PA形成1∶1复合物，灭活纤溶酶原。目前研究认为，在肺纤维化的发展过程中，PAI-1调节体内多种细胞如白细胞、纤维母细胞的黏附和移行，进入损伤组织，并与多种细胞因子TGF-β、TNF-α、PDGF等相互作用，进一步上调自己的表达，使PAI-1大量分泌，活性升高，u-PA活性降低，纤溶系统受到损害，不能完全清除已形成的纤维蛋白，从而加速肺纤维化的发生发展。虽然IPF潜在的发展机制不清楚，最新的证据表明，增加PAI-1的表达对IPF发病机制有很重要贡献。博莱霉素（Bleomycin, BLM）致肺纤维化的转基因大鼠模型证实了肺纤维化的程度与PAI-1的基因密度呈明显正相关。目前，已有应用siRNA技术沉默PAI-1表达减轻肝纤维化的报道。 体外研究表明，PAI-1可以促进肝星状细胞、血管平滑肌细胞增殖，，并抑制其凋亡。本研究中，为了探讨PAI-1在肺纤维化发生中的作用，我们将体外培养的大鼠胚肺成纤维细胞加入外源性PAI-1，观察成纤维细胞增殖、转化、胶原合成的变化。进一步通过TNF-α诱导成纤维细胞凋亡后，观察PAI-1对体外培养的大鼠胚肺成纤维细胞自发及诱导凋亡的影响及机制。 方法： 1外源性PAI-1对成纤维细胞增殖的影响及信号转导通路 将冻存的大鼠胚肺成纤维细胞复苏后，分为以下三组：Control组、PAI-1组和TGF-β组。在培养液中分别加入相应的物质使其浓度分别达到20ng/ml（PAI-1）和2ng/ml（TGF-β）。我们前期研究结果表明，这一浓度促进成纤维细胞增殖最明显。应用western blotting法分别测定24h、48h和72h PAI-1、α-SMA、AKT、p-AKT、ERK、p-ERK蛋白的表达；应用RT-PCR和Real time PCR分别测定24h的PAI-1、α-SMA、Ⅰ型胶原、Ⅲ型胶原mRNA的表达；应用激光共聚焦显微镜观察24h、48h细胞内游离Ca~(2+)浓度的变化。 2外源性PAI-1对成纤维细胞凋亡的影响及信号通路 将冻存的大鼠胚肺成纤维细胞复苏后，分为以下四组：Control组、PAI-1组、TNF-α+anti-Fas组和TNF-α+anti-Fas+PAI-1组。在培养液中分别加入相应的物质，使其在培养液中达到如下浓度：PAI-1：20ng/ml；TNF-α：20ng/ml；anti-Fas：1μg/ml，应用western blotting法测PAI-1、Caspase-3、NF-κB、AKT、p-AKT、ERK、p-ERK蛋白的表达。 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，有显著差异者用最小显著差法（least significant difference，LSD）进行两两比较，P0.05为差异有统计学意义。 结果： 1复苏后的成纤维细胞呈圆球形，约4-5小时即贴壁、开始伸展，完全伸展的细胞形状如梭形，细胞透亮，连接紧密，逐渐生长呈放射状，互相连接，复苏成功的细胞为第2代，实验用第3-5代。 2经过外源性PAI-1刺激后，可以使成纤维细胞内PAI-1在24h高表达，并持续到72h。同时，使成纤维细胞内α-SMA、Ⅰ型胶原、Ⅲ型胶原的表达、细胞内Ca~(2+)浓度、磷酸化ERK、AKT的表达增加（P0.05）。PAI-1的上述作用与TGF-β作用相似。 3外源性PAI-1使Caspase-3表达下调并抑制经TNF-α致敏、anti-Fas诱导的Caspase-3表达上调，同时上调NF-κB、磷酸化ERK和AKT蛋白的表达（P0.05）。 结论： 1PAI-1可以促进成纤维细胞增殖、转化和合成胶原，这些作用可能与PAI-1增加细胞内Ca~(2+)浓度，活化AKT、ERK信号途径有关。 2PAI-1可以抑制成纤维细胞自发凋亡及TNF-α诱发的成纤维细胞凋亡，这些作用可能与PAI-1增加细胞内Ca~(2+)浓度，活化AKT、ERK、NF-κB信号途径有关。
[Abstract]:Background and purpose: idiopathic pulmonary fibrosis (IPF) is the most common type of idiopathic interstitial pneumonia, accounting for about 47% to 71%, and its prognosis is poor. The median survival time is only 3 to 4 years, and the main pathological features of.IPF are alveolar type I epithelial cells, vascular endothelial cells and basilar membrane damage accompanied by basophilia. Inflammatory cells such as neutrophils, macrophages and lymphocytes are infiltrated, and alveolar type II epithelial cells and fibroblasts are abnormally proliferated. The basic cause of excessive collagen deposition of.IPF is unclear. In previous studies, many factors are considered to be related to the inflammatory and fibrotic processes involved in many cytokines, such as TGF - beta (TGF). TGF- beta), tumor necrosis factor - alpha (TNF- alpha), platelet derived growth factor (PDGF), matrix metalloproteinase MMP and so on.
The main function of the plasminogen activator inhibitor -1 (Plasminogen activator inhibitor-1, PAI-1) is to inhibit the urokinase type plasminogen activator (urokinase-type plasminogenactivator, uPA) and the tissue fibrinolytic activator (tissue-type plasminogenactivators, tPA). It forms a 1: 1 complex with u-PA or substances and inactivated During the development of pulmonary fibrosis, PAI-1 regulates the adhesion and migration of many cells in the body, such as leukocytes, fibroblasts, into the injured tissue, and the interaction with TGF- beta, TNF- a, PDGF, and so on, and further up-regulates the expression of self, so that the PAI-1 is secreted, the activity is elevated, and the activity of u-PA is reduced. Low, fibrinolysis system is impaired and can not completely remove the formed fibrin and accelerate the development of pulmonary fibrosis. Although the potential development mechanism of IPF is unclear, the latest evidence suggests that the increase of PAI-1 expression is very important for the pathogenesis of IPF. The transgenic rat model of bleomycin (Bleomycin, BLM) induced pulmonary fibrosis It is confirmed that the degree of pulmonary fibrosis is positively correlated with the gene density of PAI-1. At present, siRNA technology has been used to silence PAI-1 expression to alleviate liver fibrosis.
In vitro studies have shown that PAI-1 can promote the proliferation of hepatic stellate cells and vascular smooth muscle cells and inhibit its apoptosis. In this study, in order to explore the role of PAI-1 in the pathogenesis of pulmonary fibrosis, we added exogenous PAI-1 to rat embryonic lung fibroblasts cultured in vitro, and observed the changes in fibroblast proliferation, transformation, and collagen synthesis. After the apoptosis of fibroblasts was induced by TNF- alpha, the effects and mechanisms of PAI-1 on spontaneous and induced apoptosis of cultured rat embryonic lung fibroblasts were observed.
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