【作者】 王萍；

【导师】 李铁刚；

【作者基本信息】 中国医科大学 ， 急诊医学， 2020， 硕士

【摘要】 目的:百草枯(Paraquat,简称PQ)是一种广泛使用的除草剂,对人畜均有较强的毒性,且无解毒剂,PQ中毒晚期可导致肺部纤维化直至呼吸衰竭而死亡。根据中毒后肺部上皮细胞转化为间质细胞的生理过程。我们认为缺氧诱导因子(HIF)在PQ中毒导致肺纤维化中起到了重要作用。在PQ中毒导致细胞严重缺氧的条件下,HIF通过结合缺氧反应元件激活下游众多靶基因,从而参与与肺部上皮细胞的血管生成和上皮间质转化(EMT)的环节。HIF也可以直接与细胞信号通路发生反应。本文旨在探索HIF-1α通过Notch1信号通路来调节PQ中毒所致肺部EMT的机制,从而控制纤维化的发生和进展,为PQ中毒的治疗寻找潜在的靶点。方法:1、PQ中毒的小鼠模型建立:以腹腔注射方式染毒,构建PQ染毒后小鼠的肺纤维化模型。方法:A组(生理盐水对照组),B组10mg/kg(单次染毒组),C组:20mg/kg(单次染毒组),D组(10mg/kg隔日一次染毒组,共三次)染毒后观察至第21天麻醉处死小鼠取肺组织,镜下观察病理组织变化,马松染色,Ashcroft评分等方法来评估纤维化程度,提取小鼠肺组织蛋白以Western Blot检测HIF-1α和Notch1的表达情况。2、PQ中毒的细胞模型建立:体外培养的A549细胞系,以终浓度为0,10,30和60μg/ml的PQ进行染毒,染毒观察6天后,MTT法检测细胞活力,Transwell实验观察纤维化细胞的迁移能力,Western Blot和Realtime RT-PCR检测α-SMA和Ecadherin等纤维化指标,并着重检测HIF-1α和Notch1在细胞中毒模型上的蛋白和基因表达情况。3、siRNA干扰验证实验:在上述PQ中毒细胞模型的基础上,以特异性iRNA敲低HIF-1α和Notch1表达,分析α-SMA和E-cadherin的表达及EMT程度的变化,验证HIF-1α-Notch1通路的下调对PQ染毒后EMT的缓解。4、免疫共沉淀(Co-IP)实验:在上述PQ中毒细胞模型的基础上,以Co-IP分析HIF-1α和Notch1蛋白之间的直接相互作用。结果:1、在PQ中毒导致纤维化的小鼠模型中,HIF-1α和Notch1在肺组织中高表达。2、在PQ中毒A549细胞模型中,HIF-1α和Notch1在染毒后细胞中呈高表达。3、在PQ中毒细胞模型中,以特异性iRNAs敲低HIF-1α和Notch1的表达,可降低EMT标记物α-SMA的表达并升高上皮细胞标志物E-cadherin的表达,提示可能对纤维化过程有一定缓解作用。4、在PQ中毒细胞模型中,HIF-1α和Notch1分子之间可能存在直接的相互作用。结论:在小鼠在体和细胞离体水平上模拟了PQ慢性中毒诱导肺EMT致的过程,该过程可激活HIF-1α-Notch1信号通路,表现为HIF-1α和Notch1表达上调,且二者之间存在分子间直接的相互作用。而针对HIF-1α/Notch1通路进行干扰,可缓解PQ中毒所致肺细胞EMT的特征。更多还原

【Abstract】 Objective: Paraquat(PQ)is a widely used herbicide.It is highly toxic to humans and animals and has no antidote.Late PQ poisoning can lead to lung fibrosis and death from respiratory failure.According to the physiological process of lung epithelial cells transformed into interstitial cells after poisoning.We believe that hypoxia-inducible factor(HIF)plays an important role in PQ poisoning leading to pulmonary fibrosis.Under the condition that PQ poisoning causes severe cell hypoxia,HIF activates many target genes downstream by combining hypoxia response elements,thereby participating in the angiogenesis and epithelialmesenchymal transition(EMT)of lung epithelial cells.HIF can also directly react with cell signaling pathways.This article aims to explore the mechanism by which HIF-1α regulates pulmonary EMT caused by PQ poisoning through the Notch1 signaling pathway,thereby controlling the occurrence and progression of fibrosis,and finding potential targets for the treatment of PQ poisoning.Method:1.Establishment of a mouse model of PQ poisoning: Exposure by intraperitoneal injection to construct a model of pulmonary fibrosis in mice after PQ exposure.Methods: Group A saline control group,Group B 10 mg / kg(single exposure group),Group C: 20 mg / kg(single exposure group),Group D(10 mg / kg group once every other day,three times in total)observe the anesthesia and kill the mice on the 21 st day after exposure Tissue,microscopic observation of pathological changes,Masson staining,Ashcroft score and other methods to assess the degree of fibrosis,extraction of mouse lung tissue protein and Western Blot to detect the expression of HIF-1α and Notch1.2.Establishment of a cell model for PQ poisoning: A549 cell lines cultured in vitro were infected with PQ at final concentrations of 0,10,30,and 60 μg / ml.After 6 days of exposure,MTT method to detect cell viability,Transwell experiment observed the migration ability of fibrotic cells,Western Blot and Realtime RT-PCR detect α-SMA and E-cadherin and other fibrosis indicators,and focus on the detection of protein and gene expression of HIF-1α and Notch1 in cell poisoning models.3.siRNA interference verification experiment: on the basis of the above PQ poisoning cell model,knock down the expression of HIF-1α and Notch1 with specific iRNA,analyze the expression of α-SMA and E-cadherin and the change of EMT degree,verify HIF-1α-Down-regulation of Notch1 pathway alleviates EMT after PQ exposure.4.Co-IP experiment: Based on the above PQ poisoning cell model,Co-IP was used to analyze the direct interaction between HIF-1α and Notch1 protein.Results:1.In a mouse model of fibrosis caused by PQ poisoning,HIF-1α and Notch1 are highly expressed in lung tissue.2.In the PQ poisoning cell model,HIF-1α and Notch1 are highly expressed in the infected cells.3.In the model of PQ poisoning cells,knocking down the expression of HIF-1α and Notch1 with specific iRNA can reduce the expression of EMT marker α-SMA and increase the expression of epithelial cell marker E-cadherin,suggesting that The chemical process has a certain relief effect.4.In the model of PQ poisoning cells,there may be a direct interaction between HIF-1α and Notch1 molecules.Conclusion:The process of PQ-induced lung fibrosis induced by chronic PQ poisoning was simulated at the in vivo and in vitro levels in mice.This process can activate the HIF-1α-Notch1 signaling pathway,and the expression of HIF-1α and Notch1 is up-regulated,and both There is a direct interaction between the molecules.Interference with the HIF-1α / Notch1 pathway can down-regulate the characteristics of lung cell EMT caused by PQ poisoning.更多还原