本文選題：肺纖維化 + 成纖維細(xì)胞��； 參考：《河北醫(yī)科大學(xué)》2012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，約占47%～71%，其預(yù)后差，生存中位時(shí)間僅為3至4年。IPF主要病理特征為肺泡Ⅰ型上皮細(xì)胞、血管內(nèi)皮細(xì)胞及基底膜的損害，并伴有嗜中性粒細(xì)胞、巨噬細(xì)胞和淋巴細(xì)胞等炎癥細(xì)胞浸潤(rùn)，同時(shí)肺泡Ⅱ型上皮細(xì)胞與成纖維細(xì)胞異常增生，膠原過(guò)度沉積。IPF的基本病因目前還不清楚，在以往的研究中多認(rèn)為與多種細(xì)胞因子參與的炎癥和纖維化過(guò)程有關(guān)，如:轉(zhuǎn)化生長(zhǎng)因子-β(TGF-β)、腫瘤壞死因子-α(TNF-α)、血小板源性生長(zhǎng)因子(PDGF)、基質(zhì)金屬蛋白酶MMP等。 纖溶酶原激活物抑制劑-1（Plasminogen activator inhibitor-1, PAI-1）的主要作用是抑制尿激酶型纖溶酶原激活物(urokinase-type plasminogenactivator, uPA)和組織纖維蛋白溶酶原激活劑(tissue-type plasminogenactivators, tPA)，它與u-PA或t-PA形成1∶1復(fù)合物，滅活纖溶酶原。目前研究認(rèn)為，在肺纖維化的發(fā)展過(guò)程中，PAI-1調(diào)節(jié)體內(nèi)多種細(xì)胞如白細(xì)胞、纖維母細(xì)胞的黏附和移行，進(jìn)入損傷組織，并與多種細(xì)胞因子TGF-β、TNF-α、PDGF等相互作用，進(jìn)一步上調(diào)自己的表達(dá)，使PAI-1大量分泌，活性升高，u-PA活性降低，纖溶系統(tǒng)受到損害，不能完全清除已形成的纖維蛋白，從而加速肺纖維化的發(fā)生發(fā)展。雖然IPF潛在的發(fā)展機(jī)制不清楚，最新的證據(jù)表明，增加PAI-1的表達(dá)對(duì)IPF發(fā)病機(jī)制有很重要貢獻(xiàn)。博萊霉素（Bleomycin, BLM）致肺纖維化的轉(zhuǎn)基因大鼠模型證實(shí)了肺纖維化的程度與PAI-1的基因密度呈明顯正相關(guān)。目前，已有應(yīng)用siRNA技術(shù)沉默PAI-1表達(dá)減輕肝纖維化的報(bào)道。 體外研究表明，PAI-1可以促進(jìn)肝星狀細(xì)胞、血管平滑肌細(xì)胞增殖，，并抑制其凋亡。本研究中，為了探討PAI-1在肺纖維化發(fā)生中的作用，我們將體外培養(yǎng)的大鼠胚肺成纖維細(xì)胞加入外源性PAI-1，觀察成纖維細(xì)胞增殖、轉(zhuǎn)化、膠原合成的變化。進(jìn)一步通過(guò)TNF-α誘導(dǎo)成纖維細(xì)胞凋亡后，觀察PAI-1對(duì)體外培養(yǎng)的大鼠胚肺成纖維細(xì)胞自發(fā)及誘導(dǎo)凋亡的影響及機(jī)制。 方法： 1外源性PAI-1對(duì)成纖維細(xì)胞增殖的影響及信號(hào)轉(zhuǎn)導(dǎo)通路 將凍存的大鼠胚肺成纖維細(xì)胞復(fù)蘇后，分為以下三組：Control組、PAI-1組和TGF-β組。在培養(yǎng)液中分別加入相應(yīng)的物質(zhì)使其濃度分別達(dá)到20ng/ml（PAI-1）和2ng/ml（TGF-β）。我們前期研究結(jié)果表明，這一濃度促進(jìn)成纖維細(xì)胞增殖最明顯。應(yīng)用western blotting法分別測(cè)定24h、48h和72h PAI-1、α-SMA、AKT、p-AKT、ERK、p-ERK蛋白的表達(dá)；應(yīng)用RT-PCR和Real time 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)；應(yīng)用激光共聚焦顯微鏡觀察24h、48h細(xì)胞內(nèi)游離Ca~(2+)濃度的變化。 2外源性PAI-1對(duì)成纖維細(xì)胞凋亡的影響及信號(hào)通路 將凍存的大鼠胚肺成纖維細(xì)胞復(fù)蘇后，分為以下四組：Control組、PAI-1組、TNF-α+anti-Fas組和TNF-α+anti-Fas+PAI-1組。在培養(yǎng)液中分別加入相應(yīng)的物質(zhì)，使其在培養(yǎng)液中達(dá)到如下濃度：PAI-1：20ng/ml；TNF-α：20ng/ml；anti-Fas：1μg/ml，應(yīng)用western blotting法測(cè)PAI-1、Caspase-3、NF-κB、AKT、p-AKT、ERK、p-ERK蛋白的表達(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，有顯著差異者用最小顯著差法（least significant difference，LSD）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復(fù)蘇后的成纖維細(xì)胞呈圓球形，約4-5小時(shí)即貼壁、開(kāi)始伸展，完全伸展的細(xì)胞形狀如梭形，細(xì)胞透亮，連接緊密，逐漸生長(zhǎng)呈放射狀，互相連接，復(fù)蘇成功的細(xì)胞為第2代，實(shí)驗(yàn)用第3-5代。 2經(jīng)過(guò)外源性PAI-1刺激后，可以使成纖維細(xì)胞內(nèi)PAI-1在24h高表達(dá)，并持續(xù)到72h。同時(shí)，使成纖維細(xì)胞內(nèi)α-SMA、Ⅰ型膠原、Ⅲ型膠原的表達(dá)、細(xì)胞內(nèi)Ca~(2+)濃度、磷酸化ERK、AKT的表達(dá)增加（P0.05）。PAI-1的上述作用與TGF-β作用相似。 3外源性PAI-1使Caspase-3表達(dá)下調(diào)并抑制經(jīng)TNF-α致敏、anti-Fas誘導(dǎo)的Caspase-3表達(dá)上調(diào)，同時(shí)上調(diào)NF-κB、磷酸化ERK和AKT蛋白的表達(dá)（P0.05）。 結(jié)論： 1PAI-1可以促進(jìn)成纖維細(xì)胞增殖、轉(zhuǎn)化和合成膠原，這些作用可能與PAI-1增加細(xì)胞內(nèi)Ca~(2+)濃度，活化AKT、ERK信號(hào)途徑有關(guān)。 2PAI-1可以抑制成纖維細(xì)胞自發(fā)凋亡及TNF-α誘發(fā)的成纖維細(xì)胞凋亡，這些作用可能與PAI-1增加細(xì)胞內(nèi)Ca~(2+)濃度，活化AKT、ERK、NF-κB信號(hào)途徑有關(guān)。
[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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本文編號(hào)：1790724