本文選題：自噬　切入點：上皮間質(zhì)轉(zhuǎn)化　出處：《安徽醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的放療是臨床治療惡性腫瘤的常用方法,然而胸部照射誘發(fā)的放射性肺纖維化(radiation-induced pulmonary fibrosis,RPF),是放療最為棘手的并發(fā)癥。放射性肺纖維化病理上以成纖維細(xì)胞聚集,肺泡II型上皮細(xì)胞損傷,細(xì)胞外基質(zhì)和膠原沉積為主要特征。我們的前期研究表明,輻射誘導(dǎo)肺泡上皮細(xì)胞(alveolar epithelial cells,AECs)發(fā)生上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)進(jìn)而轉(zhuǎn)化成成纖維細(xì)胞是放射性肺纖維化的重要細(xì)胞來源。自噬(autophagy)是細(xì)胞在靜息和應(yīng)激狀態(tài)下維持內(nèi)環(huán)境穩(wěn)態(tài)的一種高度保守的細(xì)胞行為。通過自噬體、溶酶體的相繼組裝形成自噬溶酶體,從而實現(xiàn)胞內(nèi)需降解的細(xì)胞器、蛋白質(zhì)等成分的降解反應(yīng)。自噬的發(fā)生主要以自噬體膜結(jié)合型標(biāo)志蛋白LC3I/II、Beclin-1的誘導(dǎo)表達(dá)、p62的降解等為典型特征。在實驗室以往的研究中,我們發(fā)現(xiàn),EMT的重要分子信號通路蛋白β-連環(huán)蛋白(β-catenin)在肺組織上皮細(xì)胞胞漿和胞核中表達(dá)增加,從而促進(jìn)放射性肺纖維化中成纖維細(xì)胞增多,本研究從自噬在EMT引起放射性肺纖維化過程中的作用出發(fā),探討自噬是否通過Wnt/β-catenin通路來介導(dǎo)EMT。因此我們希望系統(tǒng)研究自噬介導(dǎo)輻射誘導(dǎo)肺泡EMT的作用機(jī)制,為探索放射性肺纖維化的發(fā)病機(jī)制及防止放射性肺纖維化的進(jìn)一步發(fā)展提供新的線索。方法以人支氣管上皮細(xì)胞(Beas-2B)為靶細(xì)胞,給予6 Gy~(60)Coγ射線照射,利用免疫印跡技術(shù)在蛋白水平檢測EMT、低氧誘導(dǎo)因子-1α(hypoxia-inducible factor-1α,HIF-1α)、自噬、Wnt/β-catenin信號通路的蛋白表達(dá)和活化情況,利用激光共聚焦技術(shù)檢測EMT表面蛋白的表達(dá)變化,利用流式分選技術(shù)分析EMT表面蛋白表型的改變。結(jié)果6Gy~(60)Coγ射線照射Beas-2B細(xì)胞48h后發(fā)生明顯的EMT,建立輻射誘導(dǎo)的EMT模型。并且發(fā)現(xiàn)照射后細(xì)胞內(nèi)HIF-1α蛋白被顯著的誘導(dǎo)表達(dá),形成誘導(dǎo)低氧微環(huán)境;同時自噬標(biāo)志性蛋白LC3I/II,Bcelin-1表達(dá)增多,而p62表達(dá)下調(diào),促使細(xì)胞自噬發(fā)生;60Coγ射線照射后引起β-catenin蛋白表達(dá)增加,當(dāng)下調(diào)β-catenin表達(dá)水平后,EMT相關(guān)蛋白表達(dá)抑制,而自噬標(biāo)志性蛋白的表達(dá)水平并未出現(xiàn)明顯改變;抑制自噬后,Wnt/β-catenin信號通路活化受到明顯抑制;與此同時EMT相關(guān)蛋白表達(dá)下調(diào)。結(jié)論60Coγ射線照射后形成低氧誘導(dǎo)微環(huán)境,誘導(dǎo)了細(xì)胞自噬反應(yīng)的發(fā)生,從而通過激活Wnt/β-catenin信號通路介導(dǎo)了支氣管上皮間質(zhì)轉(zhuǎn)化。
[Abstract]:Objective radiotherapy is a common method in the treatment of malignant tumor. However, radiation-induced pulmonary fibrosis induced by chest irradiation is the most difficult complication of radiotherapy. Injury of alveolar type II epithelial cells, extracellular matrix and collagen deposition are the main characteristics. Radiation induced alveolar epithelial cells (AECs) induced epithelial-mesenchymal transition (EMT) and then transformed into fibroblasts is an important cell source for radiation-induced pulmonary fibrosis. Autophagyis is an important cell source to maintain the homeostasis of the cells under rest and stress. A highly conserved cellular behavior that passes through autophages. The lysosomes were assembled one after another to form autophagic lysosomes, thereby realizing the cellular organelles that degraded the internal demand of the cells. Degradation of proteins and other components. Autophagy is mainly characterized by the degradation of autophagy membrane binding marker LC3I- / IIBeclin-1, which is the inducible expression of p62. We found that the expression of 尾 -catenin, an important molecular signal transduction protein of EMT, was increased in the cytoplasm and nucleus of lung epithelial cells, thus promoting the proliferation of fibroblasts in radiation-induced pulmonary fibrosis. Based on the role of autophagy in the process of radiation-induced pulmonary fibrosis induced by EMT, this study explored whether autophagy mediates EMT-mediated by Wnt/ 尾 -catenin pathway. Therefore, we hope to systematically study the mechanism of autophagy induced radiation induced alveolar EMT. In order to explore the pathogenesis of radiation-induced pulmonary fibrosis and to prevent the further development of radiation-induced pulmonary fibrosis, we used human bronchial epithelial cells (Beas-2B) as target cells and were exposed to 6 Gy~(60)Co 緯 -rays. The expression and activation of EMT, hypoxia-inducible factor-1 偽 -HIF-1 偽 and autophagy Wnt- 尾 -catenin signaling pathway were detected by Western blotting at protein level. The changes of protein expression on EMT surface were detected by confocal laser technique. The phenotypic changes of EMT surface proteins were analyzed by flow sorting technique. Results after 48 h of 6Gy~(60)Co 緯 -ray irradiation on Beas-2B cells, significant EMT-induced EMT model was established, and the expression of HIF-1 偽 protein in the cells was significantly induced after irradiation. At the same time, the expression of autophagy iconic protein LC3I / IIG Bcelin-1 was increased, while p62 expression was down-regulated, which promoted the increase of 尾 -catenin protein expression after autophagy exposed to 60Co 緯 -rays, and inhibited the expression of EMT related protein after down-regulation of 尾 -catenin expression level. However, the expression level of autophagy iconic protein was not significantly changed, the activation of Wnt- 尾 -catenin signaling pathway was significantly inhibited after autophagy, and the expression of EMT related protein was down-regulated. Conclusion 60Co 緯 -ray irradiation can induce hypoxia-induced microenvironment. Autophagy was induced, and the interstitial transformation of bronchial epithelium was mediated by activation of Wnt/ 尾 -catenin signaling pathway.
【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R730.55

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 熊珊珊;李若曦;杜麗;肖鳳君;徐龍;劉坤璐;楊陟華;朱茂祥;潘秀頡;;纖維細(xì)胞和Th1/Th2型細(xì)胞因子在放射性肺損傷中的作用及相關(guān)機(jī)制[J];中華放射醫(yī)學(xué)與防護(hù)雜志;2013年02期

相關(guān)博士學(xué)位論文 前2條

1 熊珊珊;調(diào)節(jié)性T淋巴細(xì)胞（Treg）在放射性肺纖維化中的作用及機(jī)制研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2015年

2 王寶中;IL-17A在放射性肺損傷中的表達(dá)及其作用的研究[D];山東大學(xué);2014年

相關(guān)碩士學(xué)位論文 前1條

1 黃山英;放射性肺纖維化的分子機(jī)制及防治研究[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2005年

，

本文編號：1649955