【作者】 刘佳；

【导师】 罗凤鸣；

【作者基本信息】 四川大学 ， 内科学（呼吸）， 2023， 博士

【摘要】 目的:间质性肺疾病(Interstitial lung disease,ILD)是一组累及肺间质、肺泡、细支气管、小血管和(或)淋巴管的弥漫性肺疾病,以不同形式和程度的炎症和纤维化为主要病理特征。肺纤维化过程伴随着细胞外基质过度沉积、肺泡正常结构破坏和进行性肺功能减退等。多种因素可致肺纤维化,如遗传因素、免疫因素、吸烟、环境污染、药物、职业粉尘暴露、病毒感染等。纤维化性间质性肺疾病患者预后不良。特发性肺纤维化(Idiopathic pulmonary fibrosis,IPF)是纤维化性间质性肺疾病中最典型的,IPF患者确诊后的中位生存期是3-5年。国际心肺移植学会2014年报告指出ILD患者初次肺移植术后的中位生存时间仅4.7年,再次肺移植后的中位生存时间为3.1年。吡非尼酮和尼达尼布是目前仅有的被批准用于抗肺纤维化治疗的两种药物,但两种药物仅能延缓患者肺功能的下降速度。目前仍缺乏针对肺纤维化的有效治疗措施,深入探索肺纤维化发生和发展的病理机制,挖掘潜在治疗靶点具有重要的临床和科研价值。RUNX1是Runt相关转录因子家族(Runt-related transcription factor family)成员,可通过转录调控基因表达参与多种生物学过程,如细胞增殖、分化、凋亡、自噬、衰老等等。既往RUNX1相关研究主要集中在血液系统疾病及实体肿瘤领域,近年来发现其参与调控肾脏纤维化病变,亦是心肌梗死后心肌重塑(心肌纤维化)的重要调控分子。研究表明,RUNX1是调控间充质细胞(心肌成纤维细胞、前列腺间充质干细胞等)转分化的重要蛋白,在唐氏综合症患者的心肌成纤维细胞中RUNX1更是能调控约80%的细胞外基质相关基因的表达。成纤维细胞是肺组织重塑和细胞外基质过度沉积阶段的重要效应细胞。鉴于既往研究已明确指出成纤维细胞具有高度异质性,主要表现为不同器官或组织的成纤维细胞具有独特的基因表达模式,同一器官或组织内的成纤维细胞可呈现出不同的极性。RUNX1是否能通过调控肺成纤维细胞参与肺纤维化还有待进一步解析。研究显示TGF-β、JAK-STAT等信号通路的调控紊乱是促进肺纤维化的重要原因。其他学者的研究结果提示在免疫细胞和皮肤上皮细胞中,RUNX1对TGF-β和JAK-STAT信号通路具有调控作用。因此我们提出科学假设:RUNX1很可能在肺成纤维细胞中参与调控JAK-STAT信号通路驱动肺纤维化。为验证本研究的科学假说,我们拟通过利用疾病临床样本分子表型研究、生物信息学分析、细胞和动物实验等研究手段,探究RUNX1在肺纤维化中的作用。同时,我们以肺成纤维细胞的转分化调控为研究核心,进一步探索RUNX1参与肺纤维化的细胞生物学机制,并用博来霉素(Bleomycin,BLM)诱导的肺纤维化小鼠模型,探索RUNX1抑制剂在肺纤维化预防和治疗中的作用。材料和方法:1)评估RUNX1 mRNA和RUNX1蛋白在人和小鼠纤维化肺组织中整体变化趋势。利用GEO公共数据库中的普通转录组测序数据集,探索RUNX1 mRNA(人源)在IPF肺组织内的表达情况,探索RUNX1 mRNA表达与IPF患者肺功能相关指标的相关性。利用IPF患者病肺组织病理切片进行免疫组化染色检测RUNX1蛋白水平。构建鼻滴BLM诱导的小鼠肺纤维化疾病模型,评估建模效果后,检测小鼠肺纤维化组织中RUNX1蛋白水平变化。2)评估RUNX1 mRNA和RUNX1蛋白在人和小鼠病灶成纤维细胞中的变化趋势。利用GEO公共数据库中的单细胞测序数据集,探索RUNX1 mRNA(人源)和Runx1 mRNA(鼠源)分别在IPF患者和BLM诱导的小鼠纤维化肺组织中不同细胞亚群内的表达变化趋势;利用IPF患者病肺组织病理切片及BLM诱导的小鼠肺纤维化组织病理切片,通过免疫荧光双染观测RUNX1蛋白在成纤维细胞亚群内的水平改变。3)评价RUNX1的表达异常(过表达和沉默RUNX1基因表达,抑制RUNX1蛋白功能)对人肺原代成纤维细胞转分化和细胞外基质相关蛋白水平的影响。分离和纯化人肺组织来源的原代成纤维细胞,经过3～6次传代以后进行体外实验。给予TGF-β细胞因子诱导肺成纤维细胞转分化、腺病毒载体(Adenovirus,Ad)过表达RUNX1、小干扰RNA(Small interfering RNA,siRNA)抑制RUNX1的表达,观察RUNX1对细胞外基质相关蛋白合成和成纤维细胞转分化的影响。预防性和治疗性给予Ro5-3335(RUNX1抑制剂),观察Ro5-3335对TGF-β细胞因子和腺病毒过表达RUNX1诱导的蛋白水平的影响。4)评估IPF中JAK-STAT信号通路重要角色及RUNX1参与JAK-STAT信号通路的方法文献计量学分析:通过citexs平台分析肺纤维化与JAK-STAT信号通路相关文献特征,探索JAK-STAT信号通路在肺纤维化中的重要作用。生物信息学分析方法:IPF Cell Atlas公共数据库探索JAK和STAT家族分子在IPF患者成纤维细胞中的变化趋势。蛋白互作分析RUNX1对JAK-STAT家族蛋白分子的调控情况。在肺原代成纤维细胞中过表达RUNX1,验证过表达成功后送普通转录组测序(RNA sequencing,RNA-seq)分析RUNX1调控的信号通路及生物过程。细胞学分析方法:在肺原代成纤维细胞中,体外转染RUNX1 siRNA,Western blot检测JAK-STAT信号通路相关蛋白水平的改变。5)评价RUNX1对其他肺成纤维细胞表型(增殖、迁移、衰老)的影响在肺原代成纤维细胞中,体外给予腺病毒过表达RUNX1、转染RUNX1siRNA、TGF-β细胞因子刺激联合Ro5-3335干预,采用2种及以上方法观察改变RUNX1蛋白水平或功能后对肺成纤维细胞增殖、迁移及衰老的影响。6)设计4种Ro5-3335(RUNX1抑制剂)的给药策略,评估它们对BLM诱导的小鼠疾病模型的影响。在BLM诱导的小鼠肺纤维化疾病模型中,本研究采用鼻滴20 mg/Kg法给予小鼠Ro5-3335。为观察Ro5-3335对BLM诱导的不同肺组织病变阶段以及Ro5-3335不同时点给药对BLM诱导的小鼠肺纤维化的影响,本研究设计了四种给药策略,分别简称为炎症模型组、预防模型组、早期治疗模型组、中晚期治疗模型组,收集小鼠肺组织,通过小鼠体重变化曲线、肺组织HE、Masson病理染色、纤维化评分、炎症评分、肺泡灌洗液(Bronchoalveolar lavage fluid,BALF)及Western blot等方法分析小鼠肺纤维化病变严重程度的差异。结果:1.RUNX1在IPF患者和小鼠纤维化肺组织中表达升高:RUNX1 mRNA在IPF肺组织中增高,且与IPF患者肺一氧化碳弥散量、用力肺活量和第一秒用力呼气量呈负相关性。RUNX1蛋白在IPF患者肺组织和BLM诱导的小鼠纤维化肺组织中增高。2.RUNX1在IPF患者和小鼠的纤维化肺组织的成纤维细胞中表达明显升高:IPF肺组织的成纤维细胞群中RUNX1蛋白和RUNX1 mRNA(人源)显著增加;BLM诱导的小鼠纤维化肺组织的成纤维细胞群中RUNX1蛋白和Runx1 mRNA(鼠源)都显著上调。3.RUNX1是TGF-β细胞因子致纤维化的关键蛋白:在肺成纤维细胞中,RUNX1蛋白水平与TGF-β刺激存在浓度依赖和时间依赖性,TGF-β细胞因子上调RUNX1蛋白水平、细胞外基质相关蛋白的合成及α-SMA蛋白水平。在肺成纤维细胞中抑制RUNX1(Ro5-3335)降低TGF-β细胞因子诱导的细胞外基质相关蛋白及α-SMA蛋白水平。4.肺成纤维细胞中RUNX1的致纤维化作用:在肺成纤维细胞中,利用腺病毒过表达RUNX1后,细胞外基质相关蛋白及α-SMA蛋白水平显著增加;预防性给予Ro5-3335可降低细胞外基质相关蛋白水平;治疗性给予Ro5-3335可降低细胞外基质相关蛋白及α-SMA蛋白水平;体外转染RUNX1 siRNA可降低细胞外基质相关蛋白及α-SMA蛋白水平。5.RUNX1调控JAK-STAT信号通路在成纤维细胞的转分化中发挥作用:JAK-STAT信号通路活性在IPF中显著改变;肺成纤维细胞中过表达RUNX1后的差异基因显著富集在JAK-STAT信号通路。沉默RUNX1后肺成纤维细胞的p-JAK1,p-STAT3蛋白水平增高;RUNX1是JAK-STAT信号通路的关键调控因子。6.RUNX1对其他成纤维细胞表型(增殖、迁移、衰老)的作用:沉默RUNX1对肺成纤维细胞的增殖能力无显著影响;RUNX1抑制肺成纤维细胞的迁移,促进细胞衰老。7.预防性及治疗性给予RUNX1抑制剂均可抑制BLM诱导的小鼠肺纤维化。结论:RUNX1在IPF成纤维细胞中显著上调,并且RUNX1基因表达与IPF患者肺功能呈负相关。无论是采用预防性还是治疗性给药模式,RUNX1抑制剂均可减轻BLM诱导的小鼠肺纤维化严重程度。RUNX1是成纤维细胞JAK-STAT信号通路的关键调控因子,是肺纤维化治疗具有希望的靶点。更多还原

【Abstract】 Objective:Interstitial lung disease is a group of diffuse lung diseases involving the interstitium,alveoli,fine bronchi,small blood vessels and/or lymphatics,characterized by different forms and degrees of inflammation and fibrosis.Pulmonary fibrosis is accompanied by excessive deposition of extracellular matrix,destruction of normal alveolar structure and progressive pulmonary hypofunction,and has a variety of causative factors,such as genetic factors,immune factors,smoking,environmental pollution,drugs,occupational dust exposure,viral infections,etc.Patients with fibrotic interstitial lung disease have a poor prognosis.Idiopathic pulmonary fibrosis(IPF)is the most typical subtype of fibrotic interstitial lung disease,and the median survival of patients with IPF is 3-5 years after diagnosis.The International Society for Heart and Lung Transplantation reported that the median survival time for patients with interstitial lung fibrosis was only 4.7 years after initial lung transplantation and 3.1years after retransplantation.Currently,Pirfenidone and Nintedanib are the only two drugs approved for pulmonary fibrosis that slow the rate of decline in lung capacity.However,there is still a lack of effective therapeutic methods for pulmonary fibrosis,and it is of great clinical and scientific value to explore the pathogenesis of pulmonary fibrosis and explore potential therapeutic targets for pulmonary fibrosis in depth.Runt-related transcription factor 1(RUNX1)is a member of Runt-related transcription factor family.RUNX1 is involved in various biological processes such as cell proliferation,differentiation,apoptosis,autophagy,and senescence through transcriptional regulation of gene expression.Previous studies have focused on hematological diseases and solid tumors,but recently it has been found to be involved in the regulation of renal fibrotic lesions and an important regulator of myocardial remodeling(myocardial fibrosis)after myocardial infarction.It is also suggested that RUNX1 is an important regulatory protein for the transdifferentiation of mesenchymal cells(cardiac fibroblasts,prostate mesenchymal stem cells,etc.),and in cardiac fibroblasts from patients with Down syndrome,RUNX1 has a regulatory role in the expression of about 80% of extracellular matrix-related genes.Fibroblasts are important effector cells in the phase of excessive extracellular matrix deposition and pulmonary remodeling.Given that previous studies have clearly indicated that fibroblasts are highly heterogeneous,mainly in the sense that fibroblasts in different organs or tissues have unique gene expression patterns,and fibroblasts in the same organ or tissue can exhibit different polarities.Whether RUNX1 could participate in pulmonary fibrosis by regulating lung fibroblasts remains to be further elucidated.Dysregulation of TGF-β,JAK-STAT and other signaling pathways is an important cause of pulmonary fibrosis.The results of other scholars suggested that RUNX1 has a regulatory role on TGF-β and JAK-STAT signaling pathways in immune cells and skin epithelial cells.Therefore,we raised the scientific hypothesis: RUNX1 is likely involved in regulating JAK-STAT signaling pathway in lung fibroblasts to drive lung fibrosis.To verify the scientific hypothesis,it is proposed to explore the role of RUNX1 in lung fibrosis through molecular phenotype studies of clinical samples,bioinformatics analysis,cellular and animal experiments.Meanwhile,we focus on the transdifferentiation of lung fibroblasts to further explore how RUNX1 involve in lung fibrosis.Last but not least,we used Bleomycin(BLM)-induced pulmonary fibrosis mouse model to observe the changes in the severity of lung fibrosis with preventional and therapeutic administration of RUNX1 inhibitor in the lung.Materials and Methods:(1)Exploring the overall changes in RUNX1 mRNA and RUNX1 protein content in human and mouse fibrotic lungs.To explore the altered expression of RUNX1 mRNA(human)in patients with IPF using the bulk transcriptome sequencing datasets from the GEO database,and to explore the correlation between RUNX1 mRNA expression and lung capacity-related indicators in IPF patients.Immunohistochemical staining was used to detect RUNX1 protein levels using sections of diseased lung tissues from IPF patients.A mouse pulmonary fibrosis disease model induced by nasal drops of bleomycin(BLM)was constructed,and after evaluating the modeling effect,the changes of RUNX1 protein levels in mouse fibrotic lungs were detected.(2)Exploring the changes of RUNX1 mRNA and RUNX1 protein in fibroblasts in human and mouse fibrotic lungs.Single-cell sequencing datasets from the GEO database were used to explore the expression changes of RUNX1 mRNA(human)and Runx1 mRNA(murine)within different cell subpopulations.The levels of RUNX1 protein within cell subpopulations were observed by immunofluorescence double-staining using histopathological sections of diseased lung from IPF patients and BLM-induced lung fibrosis in mice.(3)Exploring the effects of modulating RUNX1 protein levels and function after(overexpression and silencing of RUNX1 gene expression and inhibition of RUNX1 protein function)on transdifferentiation and extracellular matrix-associated protein levels in lung fibroblasts.Primary fibroblasts of human lung tissue origin were isolated and purified,after 3-6passages,cells were used for in vitro experiments.TGF-β cytokine,adenovirus(Ad)overexpression of RUNX1,and small interfering RNA(siRNA)of RUNX1 were administered to observe the alteration of extracellular matrix-associated protein levels and fibroblast transdifferentiation ability.Prophylactic and therapeutic administration of Ro5-3335(RUNX1 inhibitor)interventions were observed for the rescue of interventions such as TGF-β cytokine stimulation and overexpression of RUNX1.(4)Assessment of the important role of JAK-STAT signaling pathway in IPF and the involvement of RUNX1 in JAK-STAT signaling pathway.Bibliometric analysis: The literature features related to pulmonary fibrosis and JAKSTAT signaling pathway were analyzed by citexs platform to explore the important role of JAK-STAT signaling pathway in pulmonary fibrosis.Bioinformatics analysis: IPF Cell Atlas public database to explore the trend of JAK and STAT family molecules in fibroblasts from IPF patients.Protein interaction analysis of RUNX1 in relation to JAK-STAT signaling pathway.Overexpression of RUNX1 in primary lung fibroblasts was verified and sent for bulk RNA transcriptome sequencing(RNA-seq)to analyze the signaling pathways and biological processes regulated by RUNX1.Cytological analysis: RUNX1 siRNA was transfected in vitro in primary lung fibroblasts and Western blot was performed to detect changes in protein levels associated with the JAK-STAT signaling pathway.(5)Evaluation of the effects of RUNX1 on proliferation,migration,and senescence of lung fibroblastsIn lung primary fibroblasts,overexpression of RUNX1,transfection with RUNX1 siRNA,and TGF-β cytokine stimulation combined with Ro5-3335 intervention were given in vitro to observe the effects on proliferation,migration,and senescence of lung fibroblasts after altering RUNX1 protein levels or function using 2 or more methods.(6)Designing four dosing strategies for Ro5-3335(RUNX1 inhibitor)and evaluate their effects on BLM-induced disease models in mice.In the BLM-induced pulmonary fibrosis,Ro5-3335 was administered intranasally at20 mg/Kg.To observe the effects of Ro5-3335 on different lung tissue lesion stages induced by BLM and the effects of Ro5-3335 administered at different time points on BLM-induced pulmonary fibrosis,four dosing strategies were designed and performed in this study,which were named inflammation model,prevention model,intervention model,and treatment model.We collected mouse lung tissues,recorded mouse body weight change curve.HE and Masson pathological staining of lung tissues,alveolar lavage fluid(Bronchoalveolar lavage fluid,BALF),and Western blot was used to analyze the differences in the severity of pulmonary fibrosis lesions in mice.Results:1.RUNX1 expression was elevated in fibrotic lung tissues from IPF patients and mice.RUNX1 mRNA expression was increased in IPF patients,and its expression were negatively correlated with forced vital capacity,forced expiratory volume in one second,and diffusing capacity of the lungs for carbon monoxide in IPF patients.RUNX1 protein levels were increased in patients with IPF and BLM-induced fibrotic lungs.2.RUNX1 expression was significantly elevated in fibroblasts of fibrotic lung tissue from IPF patients and mice.RUNX1 mRNA expression was significantly increased in the fibroblast population of fibrotic lungs from IPF and Runx1 mRNA significantly upregulated in the fibroblast cluster of BLM-induced pulmonary fibrosis.RUNX1 protein was increased in the fibroblasts of patients with IPF and BLM-induced mouse fibrotic lungs.3.RUNX1 is a key protein in TGF-β cytokine-induced fibrosis.In human lung primary fibroblasts,RUNX1 protein levels were concentration-dependent and timedependent with TGF-β cytokine stimulation.TGF-β cytokine upregulates RUNX1 protein levels,extracellular matrix-associated protein synthesis and α-SMA protein levels.Inhibition of RUNX1(Ro5-3335)in lung fibroblasts attenuated TGF-βcytokine-induced extracellular matrix-associated protein and α-SMA protein levels.4.Fibrogenic effects of RUNX1 in lung fibroblasts.In vitro,overexpressing RUNX1 significantly increased the levels of extracellular matrix-associated protein and α-SMA protein in lung fibroblasts.Prophylactic administration of Ro5-3335 reduced the expression of extracellular matrix-associated protein.Therapeutic administration of Ro5-3335 also reduced the expression of extracellular matrix-associated protein and α-SMA protein.In vitro,transfection of RUNX1 siRNA reduced the expression of extracellular matrix-associated protein and α-SMA protein.RUNX1 siRNA reduced the expression of extracellular matrix-associated protein and α-SMA protein.5.JAK-STAT signaling pathway activity is significantly altered in IPF.Differential genes after overexpression of RUNX1 in lung fibroblasts are significantly enriched in JAK-STAT signaling pathway.P-JAK1,p-STAT3 protein levels were increased in lung fibroblasts after RUNX1 silencing.RUNX1 may be involved in pulmonary fibrosis by regulating extracellular matrix synthesis through the JAK-STAT signaling pathway.6.Role of RUNX1 on other fibroblast phenotypes,such as proliferation,migration,and senescence.Silencing of RUNX1 had no significant effect on the proliferation of lung fibroblasts.RUNX1 inhibited the migration of lung fibroblasts and promoted cellular senescence.7.Both prophylactic and therapeutic administration of RUNX1 inhibitors inhibited BLM-induced pulmonary fibrosis in mice.Conclusion:RUNX1 was significantly upregulated in IPF fibroblasts,and RUNX1 mRNA expression was negatively correlated with lung capacity in patients with IPF.RUNX1 inhibitors reduce the severity of BLM-induced pulmonary fibrosis in mice,whether administered prophylactically or therapeutically.RUNX1 is a key regulator of the JAK-STAT signaling pathway in fibroblasts and a promising target for pulmonary fibrosis treatment.更多还原