【作者】 朱勇；

【导师】 王瑞兰；

【作者基本信息】 上海交通大学 ， 急诊医学， 2018， 博士

【摘要】 背景和目的:百草枯(PQ)中毒诱导的不可逆的肺纤维化是患者死亡的主要原因。上皮-间质转化(EMT)在肺纤维化的形成中发挥重要作用。本研究探讨缺氧诱导因子-1α(HIF-1α)及其靶基因micro RNA-210(miR-210)在PQ中毒致肺纤维化中对EMT的作用,及HIF-1α和miR-210之间的相互调控关系。方法:将SD大鼠随机分为对照组和模型组,模型组按50 mg/kg给予PQ一次性灌胃,并按中毒后不同时间点(2、6、12、24、48和72 h)处死分为六个亚组。体外模型应用A549和RLE-6TN细胞,分别抑制或过表达HIF-1α和miR-210,并给予相应终浓度PQ处理24 h。收集细胞和大鼠肺组织,western blot、q RT-PCR及免疫共沉淀等技术检测各指标变化。结果:PQ中毒后,体内外模型中均有上皮标志物ZO-1和E-cadherin表达减少,间质标志物α-SMA明显增多,即发生了EMT。PQ中毒2 h大鼠肺组织即有HIF-1α表达显著升高,一直到72 h均高于对照组。A549和RLE-6TN细胞内沉默HIF-1α后,上皮标志物减少明显抑制,间质标志物增多减少,细胞形态变化显著改善,Snail和β-catenin表达明显减少,PQ中毒诱导的EMT程度减轻。PQ染毒6 h大鼠肺组织miR-210明显增加,肺泡上皮细胞内抑制HIF-1α后,miR-210明显下降。体外过表达miR-210后,间质标志物α-SMA进一步增多,上皮标志物ZO-1和E-cadherin进一步减少,EMT程度加重,HIF-1α表达较PQ组也明显增多,而抑制miR-210后,明显减轻PQ中毒诱导的EMT,HIF-1α显著减少。q RT-PCR结果显示,上调或下调miR-210后,HIF-1αm RNA均无明显变化。过表达miR-210后,明显抑制RUNX3表达,HIF-1α羟化减少,与RUNX3结合的PHD2也减少,抑制miR-210后则结果相反。荧光素酶报告实验证实RUNX3是miR-210的靶基因之一。A549细胞内过表达RUNX3后,OH-HIF-1α增多,HIF-1α明显下降,抑制RUNX3后则结果相反,且PHD2的表达均无明显变化。结论:本研究明确了EMT参与PQ中毒诱导的肺纤维化,HIF-1α可能通过Snail和β-catenin途径调控EMT。PQ中毒后miR-210明显增加,并受HIF-1α调控,miR-210可促进EMT。HIF-1α和miR-210之间存在正反馈调控,miR-210可能通过抑制RUNX3,PHD2羟化HIF-1α能力下降,细胞内HIF-1α增多,一方面调节EMT,另一方面进一步促进miR-210表达,形成恶性循环,不断促进PQ诱导的EMT和肺纤维化发生发展。本研究提示HIF-1α可能是治疗PQ中毒致肺纤维化的潜在靶点。更多还原

【Abstract】 Background and objective: The irreversible pulmonary fibrosis induced by paraquat(PQ)poisoning is the main cause of death in patients.Epithelial-mesenchymal transition(EMT)plays an important role in the formation of pulmonary fibrosis.This study was designed to investigate the effect of hypoxia-inducible factor-1α(HIF-1α)and its target gene micro RNA-210(mi R-210)on EMT in PQ-induced pulmonary fibrosis,and the relationship between HIF-1α and mi R-210.Methods: The SD rats were randomly divided into control group and model group.The model group was administered with intragastric infusion of PQ at a dose of 50 mg/kg once.The rats were sacrificed at different time points(2,6,12,24,48 and 72 h).The executions were divided into six subgroups.In vitro,A549 and RLE-6TN cells were used to inhibit or overexpress HIF-1α and mi R-210,respectively,and treated with the corresponding final concentration of PQ for 24 h.Collected cells and rat lung tissue,western blot,q RT-PCR,co-immunoprecipitation and other techniques were used to detect changes.Results: After PQ poisoning in vitro and in vivo models,epithelial markers(ZO-1 and E-cadherin)expression decreased,and the interstitial marker(α-SMA)increased significantly,EMT occurred.At 2 h after PQ poisoning,the expression of HIF-1α in lung tissue of rats was significantly increased and was higher than that in the control group at 72 h.The silencing of HIF-1α in A549 and RLE-6TN cells significantly reduced the expression of epithelial markers,decreased the number of interstitial markers,significantly improved the morphological changes of cells,reduced the expression of Snail and β-catenin,and attenuated the EMT induced by PQ.The mi R-210 in lungs of rats exposed to PQ for 6 h significantly increased,and mi R-210 decreased significantly after inhibiting HIF-1α in alveolar epithelial cells.After overexpression of mi R-210 in vitro,the expression of α-SMA was further increased,the expressions of epithelial markers(ZO-1 and E-cadherin)were further decreased,the degree of EMT was increased,the expression of HIF-1α was significantly increased.After inhibiting mi R-210,significantly reduced PQ-induced EMT and HIF-1α.q RT-PCR results showed that there was no significant change in HIF-1α m RNA after mi R-210 was up-regulated or down-regulated.After overexpression of mi R-210,the expression of RUNX3 was significantly inhibited,the hydroxylation of HIF-1α was decreased,PHD2 bound to RUNX3 was also decreased,and the result was reversed after mi R-210 was inhibited.Luciferase assay confirmed that RUNX3 is one of the target genes of mi R-210.After RUNX3 was over-expressed in A549 cells,OH-HIF-1α increased and HIF-1α decreased.After inhibiting RUNX3,the results were reversed,and there was no significant change in the expression of PHD2.Conclusion: This study identified that EMT is involved in PQ poisoning-induced pulmonary fibrosis,and that HIF-1α may regulate EMT via the Snail and β-catenin pathways.After PQ poisoning,mi R-210 was significantly increased,and was regulated by HIF-1α.mi R-210 can promote PQ-induced EMT.There is a positive feedback regulation between HIF-1α and mi R-210.Mi R-210 may reduce the hydroxyapatite-HIF-1α ability of PHD2 by inhibiting RUNX3,and increase the intracellular HIF-1α,regulating EMT on the one hand and further promoting mi R-210 expression,forming a vicious circle,continue to promote PQ-induced EMT and pulmonary fibrosis development.This study suggests that HIF-1α may be a potential target for PQ poisoning-induced pulmonary fibrosis.更多还原