自噬在辐射诱导肺上皮间质转化中的作用机制研究

发布时间：2018-03-22 18:57

本文选题：自噬　切入点：上皮间质转化　出处：《安徽医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的放疗是临床治疗恶性肿瘤的常用方法,然而胸部照射诱发的放射性肺纤维化(radiation-induced pulmonary fibrosis,RPF),是放疗最为棘手的并发症。放射性肺纤维化病理上以成纤维细胞聚集,肺泡II型上皮细胞损伤,细胞外基质和胶原沉积为主要特征。我们的前期研究表明,辐射诱导肺泡上皮细胞(alveolar epithelial cells,AECs)发生上皮间质转化(epithelial-mesenchymal transition,EMT)进而转化成成纤维细胞是放射性肺纤维化的重要细胞来源。自噬(autophagy)是细胞在静息和应激状态下维持内环境稳态的一种高度保守的细胞行为。通过自噬体、溶酶体的相继组装形成自噬溶酶体,从而实现胞内需降解的细胞器、蛋白质等成分的降解反应。自噬的发生主要以自噬体膜结合型标志蛋白LC3I/II、Beclin-1的诱导表达、p62的降解等为典型特征。在实验室以往的研究中,我们发现,EMT的重要分子信号通路蛋白β-连环蛋白(β-catenin)在肺组织上皮细胞胞浆和胞核中表达增加,从而促进放射性肺纤维化中成纤维细胞增多,本研究从自噬在EMT引起放射性肺纤维化过程中的作用出发,探讨自噬是否通过Wnt/β-catenin通路来介导EMT。因此我们希望系统研究自噬介导辐射诱导肺泡EMT的作用机制,为探索放射性肺纤维化的发病机制及防止放射性肺纤维化的进一步发展提供新的线索。方法以人支气管上皮细胞(Beas-2B)为靶细胞,给予6 Gy~(60)Coγ射线照射,利用免疫印迹技术在蛋白水平检测EMT、低氧诱导因子-1α(hypoxia-inducible factor-1α,HIF-1α)、自噬、Wnt/β-catenin信号通路的蛋白表达和活化情况,利用激光共聚焦技术检测EMT表面蛋白的表达变化,利用流式分选技术分析EMT表面蛋白表型的改变。结果6Gy~(60)Coγ射线照射Beas-2B细胞48h后发生明显的EMT,建立辐射诱导的EMT模型。并且发现照射后细胞内HIF-1α蛋白被显著的诱导表达,形成诱导低氧微环境;同时自噬标志性蛋白LC3I/II,Bcelin-1表达增多,而p62表达下调,促使细胞自噬发生;60Coγ射线照射后引起β-catenin蛋白表达增加,当下调β-catenin表达水平后,EMT相关蛋白表达抑制,而自噬标志性蛋白的表达水平并未出现明显改变;抑制自噬后,Wnt/β-catenin信号通路活化受到明显抑制;与此同时EMT相关蛋白表达下调。结论60Coγ射线照射后形成低氧诱导微环境,诱导了细胞自噬反应的发生,从而通过激活Wnt/β-catenin信号通路介导了支气管上皮间质转化。
[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 纬 -rays. The expression and activation of EMT, hypoxia-inducible factor-1 伪 -HIF-1 伪 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 纬 -ray irradiation on Beas-2B cells, significant EMT-induced EMT model was established, and the expression of HIF-1 伪 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 纬 -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 纬 -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.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R730.55

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