肺部感染继发脓毒症差异基因的表达及生物信息学分析

Differential gene expression and bioinformatics analysis in sepsis secondary to pneumonia

目的通过生物信息学分析筛选肺部感染继发脓毒症的关键基因,为该疾病的基础研究提供理论依据,并寻找理想的动物模型方案。方法①实验1(生物信息学分析):利用基因表达数据库(GEO)筛选肺部感染继发脓毒症和多种脓毒症动物模型数据集,利用R软件对各数据集进行基因差异分析,差异基因进行基因本体(GO)分析和京都基因与基因组百科(KEGG)富集分析。肺部感染继发脓毒症数据集差异基因与临床症状进行相关性分析,绘制差异基因与临床症状相关性热图,利用加权基因共表达网络分析(WGCNA)和蛋白-蛋白互作用网络(PPIN)分析进行聚类筛选核心基因和关键基因。②实验2(脓毒症动物模型验证):选择体质量21~25 g的雄性小鼠,随机分为模型组和对照(Sham)组,采用盲肠结扎穿孔术(CLP)制备小鼠脓毒症损伤模型,Sham组仅需暴露盲肠。术后24 h处死小鼠取肺组织提取总RNA,用实时荧光定量聚合酶链反应(RT-qPCR)检测关键基因的mRNA表达。结果①实验1:肺部感染继发脓毒症数据集GSE 134364和GSE 65682分析后得到共有差异基因319个,其中,社区获得性肺炎(CAP)与医院获得性肺炎(HAP)在基因水平上无差异;各动物模型基因差异较大,无共有差异基因;患者与动物模型差异基因在GO功能上有相似的富集,主要在细胞分化、细胞过程的调控、细胞对刺激反应的调节等方面;KEGG通路富集的比较则差异较大,其中,CLP动物模型与患者呈现更高的一致性;WGCNA和PPIN分析后得到的关键基因有MAPK14、NLRC4和LCN2。②实验2:动物实验结果显示,CLP模型小鼠肺组织细胞中MAPK14、NLRC4和LCN2的mRNA表达较Sham组呈明显上调,与GEO的分布结果一致。结论MAPK14、NLRC4和LCN2是肺部感染继发脓毒症的关键基因,其参与了生物学过程的调节,是潜在的研究方向;CLP动物模型更能反映肺部感染继发脓毒症患者的生物学特征,是理想的动物模型方案。

Objective To analyze and screen the key genes of sepsis secondary to pulmonary infection by bioinformatics,and to provide theoretical basis for the basic research of the disease and find an ideal animal model program.Methods Experiment 1(bioinformatics analysis):gene expression data sets of pulmonary infection secondary sepsis patients and multiple sepsis animal models were screened by Gene Expression Omnibus(GEO)Database,and gene differences were analyzed by R software.Differential genes were analyzed by gene ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis.Correlation analysis was conducted between differential genes and clinical symptoms in the data set of pulmonary infection secondary sepsis,and the correlation heat map between differential genes and clinical symptoms was drawn.Key genes were screened by weighted gene co-expression network analysis(WGCNA)and protein-protein interaction network analysis(PPIN)clustering.Experiment 2(sepsis animal model preparation):male mice weighing 21-25 g were randomly divided into the key genes group and the control(Sham)group.And cecal ligation and puncture(CLP)was used to establish mouse sepsis model,while the mice in sham group were performed by exposure of cecum.And all the mice were scarified 24 hours after surgery to extract the total RNA from lung tissue,real time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect mRNA expression of key genes.Results Experiment 1(bioinformatics analysis):319 differential genes were showed by GSE 134364 and GSE 65682 data set analysis of pulmonary infection secondary sepsis.And there was no genetic difference between community acquired pneumonia(CAP)and hospital acquired pneumonia(HAP)in patients with pulmonary infection secondary to sepsis.Obvious differences existed between differential genes in animal models,and there was no common differential gene.Differential genes in patients and animal models were similarly enriched in GO function,mainly in cell differentiation,regulation of cell process,and regulation of cellular response to stimuli,there were significant differences in pathway enrichment,among which,CLP animal models showed higher consistency with patients.The key genes obtained by WGCNA and PPIN analysis were MAPK14,NLRC4 and LCN2.Experiment 2(sepsis animal model preparation):animal experiment results showed that the mRNA expressions of MAPK14,NLRC4 and LCN2 in lung tissue of CLP model mice were significantly up-regulated compared with the sham group.Conclusions MAPK14,NLRC4 and LCN2 are key genes involved in the regulation of biological processes of pulmonary sepsis secondary to infection,and are potential research directions of this disease.What's more,CLP animal model can better reflect the biological characteristics of patients with pulmonary infection secondary sepsis,and is one of the ideal animal model schemes for pulmonary infection secondary sepsis.