【作者】 廖毅；

【导师】 文富强；

【作者基本信息】 四川大学 ， 内科学（呼吸病学）（专业学位）， 2023， 博士

【摘要】 目的:特发性肺纤维化(idiopathic pulmonary fibrosis,IPF)是一种慢性、进展性的间质性肺炎,好发于中老年人群,患者常常因为持续性咳嗽以及呼吸困难而就诊。IPF起病隐匿,但预后通常较差,IPF确诊后的平均生存期约3年左右。IPF病因不明,发病机制目前也存在争议。有研究发现某些异常表达的基因以及信号通路和纤维化疾病有关,因此,本次的研究主要是探索并验证和IPF致病相关的新基因以及相关信号通路。材料和方法:1.从GEO(gene expression omnibus)数据库中寻找和IPF相关的测序数据集,利用生物信息学分析探索潜在的核心基因以及对应的信号通路,同时探究该基因对于IPF诊断以及预后判断的能力。(1)使用加权基因网络共表达(Weighted correlation network analysis,WGCNA)的方法从符合纳入标准的数据集中筛选出和IPF最为相关的模块并确定模块所对应的基因集,并进一步构建核心基因的蛋白互作网络(Protein-Protein Interaction Networks,PPI network),使用MCODE(Molecular Complex Detection)插件进一步明确靶基因。(2)对核心基因进行基因本体(gene ontology,GO)以及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析以明确和IPF相关的功能信号通路。2.Meta分析。从文献数据库寻找与IPF相关的靶基因以及其和临床相关性的文献,并利用GEO数据库中与IPF患者有关的靶基因的测序数据以及相应患者的临床信息进行诊断性以及预后性的个体病例数据的meta分析以明确靶基因的临床应用效果。3.体外实验。细胞实验以小鼠的原代成纤维细胞作为研究对象,利用外源性的SPP1以及p27激动剂丝裂霉素(Mitomycin C,MMC),PI3K抑制剂LY294002处理细胞并将其分为五组,分别是SPP1处理组,MMC处理组,LY294002处理,SPP1+MMC处理组,SPP1+LY294002处理组。将每一组细胞培养相同时间后采用western blot(WB)检测相关指标的表达程度。4.体内实验。应用si RNA将小鼠的靶基因的表达量降低。把8周龄雄性C57BL/6小鼠(体重≈20g/只)随机分为空白对照组、肺纤维化组、肺纤维化+si SPP1组。采用对小鼠气管内滴注博来霉素(Bleomycin,BLM)的方法促进小鼠的肺纤维化(0.5 mg/mL,每只小鼠使用大约100μL),对照组则采取气管内滴注无菌生理盐水的方式。我们根据造模后第7、14、28天这三个时间段将这些小鼠再次分组一共分为了9组。小鼠肺组织进行苏木精-伊红染色法(hematoxylin-eosin staining,HE)以及masson染色,并根据masson染色结果计算ashcroft肺纤维化评分。同时使用Real-time quantitative polymerase chain reaction(RT-qPCR)以及WB检测各组小鼠肺组织α-SMA,COL-1,COL-3,PI3K,p-PIK,AKT,p-AKT,p27的表达量。结果:1.WGCNA一共发现了9个模块,其中和IPF相关性最大的模块是粉色模块,该模块一共包含了287条基因,通过构建PPI并使用MCODE插件进行计算后我们发现SPP1或许和IPF的发病有关,并将其作为我们本次研究的靶基因。GO和KEGG分析发现了一些和IPF致病相关的生物学功能以及信号通路,其中包括了PI3K/AKT信号通路。我们将该PI3K/AKT作为本次研究的信号通路并通过后续的实验进行证明。2.通过对国内外数据库的仔细搜索,我们并未发现可以利用的有关SPP1用和IPF患者临床应用方面的相关文献。不过,从GEO数据库中我们获得了足够的数据。根据诊断性meta分析的结果来看,SPP1在区分IPF患者和健康对照组以及区分IPF患者和肺癌患者这两方面都表现出了极高的诊断效能。预后性meta分析提示,SPP1的高表达与不良预后相关。并且,根据来自GSE70866数据集的分析结果来看,SPP1是IPF患者独立于年龄、性别、地区的预后危险因素。3.细胞实验提示增加外源性SPP1可促进细胞纤维化,表现为α-SMA,COL-1,COL-3表达上调。p27的过表达可以减弱SPP1的促纤维化作用,SPP1+MMC组的α-SMA,COL-1,COL-3的表达量较SPP1组降低。PI3K抑制剂LY294002可以阻断SPP1对下游的调控作用。SPP1+LY294002组α-SMA,COL-1,COL-3的表达量较SPP1组减低,而p27表达量升高。4.动物实验提示通过siRNA介导成功实现肺纤维化小鼠SPP1表达下调,使用HE、Masson染色证实了除对照组外的小鼠肺组织已经出现纤维化改变。当SPP1表达下调时小鼠肺组织RT-qPCR结果提示α-SMA,COL-1,COL-3的表达量都明显降低,而p27的表达量显著升高,WB结果提示在小鼠肺组织α-SMA,COL-1,COL-3,p-PI3K,p-AKT表达量显著升高,p27表达显著降低。结论:SPP1在IPF患者中高表达,检测SPP1的表达量或许可以用于IPF患者的诊断,并且高表达SPP1的IPF患者预后相对较差。SPP1可以通过激活PI3K/AKT通路同时调控p27的表达而促进肺纤维化。更多还原

【Abstract】 Objective:Idiopathic pulmonary fibrosis(IPF)is a chronic and progressive interstitial lung disease that occurs in middle-aged and elderly people.Patients often seek medical treatment because of persistent cough and dyspnea.The onset of IPF is insidious,and the disease usually worsens gradually,and it can also manifest as acute exacerbation.The prognosis of IPF is usually poor,and the average survival period after diagnosis of IPF is about 3 years.The etiology of IPF is unknown,and the pathogenesis is still controversial.Some studies have found that some abnormally expressed genes and signaling pathways are related to fibrotic diseases.Therefore,this study is mainly to explore and verify the genes and related signaling pathways related to the pathogenesis of IPF.Materials and Methods:1.Sequencing data sets related to IPF were searched from the gene expression omnibus(GEO)database,and the potential core genes and corresponding signaling pathways were explored by bioinformatics analysis,as well as the ability of this gene for the diagnosis and prognosis of IPF.(1)Weighted gene coexpression network analysis(WGCNA)was used to select the modules most associated with IPF from the data sets meeting the inclusion criteria,and to determine the gene sets corresponding to the modules.Further,Protein-Protein Interaction Networks(PPI networks)of core genes were constructed,and MCODE(Molecular Complex Detection)plug-in was used to further identify target genes.(2)gene ontology(GO)analysis of the core genes and Kyoto encyclopedia of genes and genomes(KEGG)analysis were conducted to clarify the functional signal pathways related to IPF.2.Meta analysis.Target genes related to IPF and their clinical relevance were searched from the literature database,and the sequencing data of target genes related to IPF patients in GEO database and clinical information of corresponding patients were used for meta-analysis of individual case data of diagnosis and prognosis to clarify the clinical application effects of target genes.3.In vitro experiment.Primary mouse fibroblasts were treated with exogenous SPP1,the p27 agonist Mitomycin C(MMC)and the PI3 K inhibitor LY294002.The cells were divided into five groups: SPP1 treatment group,MMC treatment group,LY294002 treatment group.SPP1+MMC group,SPP1+LY294002 group.After incubating each group of cells for the same time,western blot(WB)was used to detect the expression degree of related indicators.4.In vivo.Mouse target gene expression was knocked down using si RNA.Clean grade 8-week-old male C57BL/6 mice(weight ≈20g/ mouse)were divided into blank control group,pulmonary fibrosis group and pulmonary fibrosis +si SPP1 group by random number table method.The pulmonary fibrosis model of mice was established by intratracheal infusion of bleomycin(0.5 mg/mL,about 100 μL per mouse),and the control group by intratracheal infusion of sterile normal saline.These mice were regrouped into 9 groups according to the time periods 7,14 and 28 days after modeling.hematoxylin-eosin staining(HE)and masson staining were performed on mouse lung tissues,and ashcroft pulmonary fibrosis score was calculated according to the results of masson staining.Real-time quantitative polymerase chain reaction(RT-qPCR)and WB were used to detect the expression ofα-SMA,COL-1,COL-3,PI3 K,p-PIK,AKT,p-AKT and p27.Results:1.A total of 9 modules were found in WGCNA,among which the pink module with the greatest correlation with IPF was the pink module,which contained 287 genes in total.After using PPI construction and MCODE plug-in for calculation,we determined that SPP1 might be related to the onset of IPF and serve as the target gene of this study.GO and KEGG identified several biological functions and signaling pathways associated with IPF pathogenesis,including PI3K/AKT signaling pathway.We took the PI3K/AKT as the signaling pathway in this study and proved it through subsequent experiments.2.Through a careful search of domestic and foreign databases,we did not find relevant literature that could be used.However,we have enough data from the GEO database.In terms of diagnostic meta-analysis,according to the results of diagnostic meta-analysis,SPP1 showed high diagnostic efficacy in distinguishing IPF patients from healthy controls and IPF patients from lung cancer patients.Prognostic meta-analysis suggested that high expression of SPP1 represented a poor prognosis.Moreover,according to the analysis results from the GSE70866 data set,SPP1 is a prognostic risk factor for IPF patients independent of age,sex,and region.3.Cell experiments suggested that increasing exogenous SPP1 could promote cell fibrosis,which was manifested as up-regulated expression of α-SMA,COL-1,and COL-3.The overexpression of p27 can weaken the profibrotic effect of SPP1,and the expressions of α-SMA,COL-1 and COL-3 in SPP1+MMC group are lower than those in SPP1 group.PI3 K inhibitor LY294002 can block the downstream regulation of SPP1.The expressions of α-SMA,COL-1 and COL-3 in SPP1+LY294002 group were decreased compared with those in SPP1 group,while the expression of p27 was increased.4.Animal experiments suggested that the expression of SPP1 in mice with pulmonary fibrosis was successfully down-regulated by siRNA mediated,and bleomycin induced pulmonary fibrosis in mice was demonstrated by HE and Masson staining.When the expression of SPP1 was down-regulated,the RT-qPCR results indicated that the expression levels of α-SMA,COL-1,and COL-3 were significantly decreased,while the expression levels of p27 were significantly increased.WB results indicated that the expression levels of α-SMA,COL-1,COL-3,p-PI3 K,and p-AKT were significantly increased.p27 expression was significantly decreased.Conclusion:SPP1 is highly expressed in IPF patients,and detecting the expression level of SPP1 may be used for the diagnosis of IPF patients,and the prognosis of IPF patients with high SPP1 expression is relatively poor.SPP1 can promote pulmonary fibrosis by activating the PI3K/AKT pathway and simultaneously regulating the expression of p27.更多还原