基于GEO数据库脓毒症心肌巨噬细胞基因芯片数据的生物信息学分析及关键基因验证

Bioinformatics analysis and key gene verification of sepsis myocardial macrophage microarray data based on GEO database

目的:利用生物信息学分析筛选脓毒症心脏组织巨噬细胞差异表达基因(DEGs)并对关键基因进行验证。方法:实验1(基因芯片与生物信息学分析):从基因表达数据库(GEO)下载脓毒症小鼠心脏巨噬细胞基因芯片数据GSE104342,通过R语言分析获得DEGs,使用DAVID在线数据库对DEGs进行基因本体及功能注释和京都基因与基因组百科全书(KEGG)富集分析,最后利用基因与蛋白质相互作用检索数据库(STRING)对DEGs进行基因编码蛋白质-蛋白质相互作用分析,并运用Cytoscape软件及MCODE等插件筛选出关键基因。实验2(脓毒症模型构建与相关基因验证):8~14周龄雄性C57BL/6小鼠10只,随机选取5只作为对照组,5只在体构建小鼠脓毒症模型作为脓毒症组。超声心动图检测小鼠心脏功能,采用苏木精-伊红染色法检测小鼠心脏形态,原位缺口末端标记法检测小鼠心肌细胞凋亡,免疫荧光染色检测分化抗原簇206(CD206)、诱导型一氧化氮合酶(iNOS)、F4/80、细胞因子信号转导抑制因子3(Socs3)、白细胞介素1受体拮抗剂(Il1rn)和CC趋化因子7(Ccl7)蛋白表达。体外培养RAW264.7巨噬细胞,分为2组:(1)脂多糖刺激组:给予1 mg/L脂多糖干预;(2)空白对照组:加入等体积磷酸缓冲液溶液。采用实时荧光定量反转录聚合酶链式反应检测巨噬细胞Socs3、Il1rn和Ccl7基因表达。结果:实验1:GSE104342数据集筛选出24647个基因,DEGs共177个(0.72%),其中上调基因120个,下调基因57个。基因本体富集分析表明,DEGs主要参与炎症反应、免疫应答、凋亡调控和抗原加工提呈等过程。KEGG信号通路分析发现,脓毒症小鼠心脏巨噬细胞中DEGs主要富集于细胞因子-细胞因子受体相互作用、肿瘤坏死因子信号通路、NOD样受体信号通路等。STRING及Cytoscape分析构建基因相互作用网络图及重要子模块,获得了3个hub基因,分别为Socs3、Il1rn和Ccl7。实验2:在体实验发现,与对照组相比,脓毒症组小鼠心脏功能明显下降,心肌细胞明显水肿、炎性细胞浸润、心肌纤维断裂,部分心肌细胞核溶解消失,心肌细胞凋亡增加,提示小鼠脓毒症心肌损伤模型构建成功。与对照组相比,脓毒症组小鼠心脏中CD206蛋白表达下调,iNOS、F4/80、Socs3、Il1rn和Ccl7蛋白表达上调,且Socs3、Il1rn、Ccl7与F4/80蛋白存在共定位。体外实验发现,与空白对照组相比,脂多糖刺激巨噬细胞后Socs3、Il1rn和Ccl7水平明显上调。结论:脓毒症小鼠心脏中巨噬细胞Socs3、Il1rn和Ccl7基因表达上调,Socs3、Il1rn和Ccl7有望成为诊治脓毒症心脏损伤的新靶点。

ObjectiveBioinformatics analysis was used to screen differentially expressed genes(DEGs)in macrophages of sepsis myocardial injury and to verify key genes.MethodsExperiment 1(gene chip and bioinformatics analysis):The gene chip data GSE104342 of cardiac macrophages in septic mice was downloaded from Gene Expression Omnibus database.DEGs were obtained by R language analysis.DAVID online database was used to obtain gene ontology and kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis of DEGs.STRING online database was used for protein‐protein interaction network analysis of DEGs,and then key genes were screened by using Cytoscape software and molecular complex detection(MCODE)plug-ins.Experiment 2(sepsis model construction and related protein verification):Ten male C57BL/6 mice,aged 8-14 weeks.Five mice were randomly selected as control group,and 5 mice were selected as the sepsis group by building a mice sepsis model in vivo.Echocardiography was used to detect the cardiac function.Hematoxylin-eosin staining was used to assess the cardiac morphology.TUNEL staining was used to evaluate cardiomyocyte apoptosis.Immunofluorescence staining was used to detect the expression of differentiation antigen cluster 206(CD206),inducible nitric oxide synthases(iNOS),F4/80,suppressor of cytokine signaling 3(Socs3),interleukin 1 receptor antagonist(Il1rn)and chemokine C-C motif ligand 7(Ccl7)protein.RAW264.7 macrophages were cultured in vitro and divided into 2 groups:LPS groupstimulated by lipopolysaccharide(LPS,1 mg/L)and blank control group treated with equal-volume phosphate buffer solution.Reverse transcription-polymerase chain reaction(RT-PCR)was used to evaluate the expression of Socs3,Il1rn and Ccl7in vitro.ResultsExperiment 1:24647 genes were screened in GSE104342 dataset and 177 genes(0.72%)were differential expression,including 120 up-regulated genes and 57 down-regulated genes.Gene ontology enrichment analysis showed that DEGs were mainly involved in inflammatory response,immune response,apoptosis regulation and antigen processing and presentation.KEGG signaling pathway analysis showed that DEGs in cardiac macrophages of septic mice were mainly enriched in cytokine-cytokine receptor interaction,tumor necrosis factor signaling pathway,NOD like receptor signaling pathway.Three hub genes were obtained by STRING and Cytoscape analysis,including Socs3,Il1rn and Ccl7.Experiment 2:In vivo,it was found that compared with the control group,the cardiac function of the sepsis mice decreased significantly,the myocardial cells were significantly edema,inflammatory cell infiltration,myocardial fiber rupture,some myocardial nuclei dissolved and disappeared,and the cardiomyocyte apoptosis increased,suggesting that the sepsis myocardial injury model of mice was successfully constructed.Compared with the control group,the expression of CD206 in the myocardium of septic mice was down-regulated,the expression of iNOS,F4/80,Socs3,Il1rn and Ccl7 were up-regulated.In addition,there was co-localization between Socs3,Il1rn,Ccl7 and F4/80 protein.Compared with the blank control group,the expression of Socs3,Il1rn and Ccl7 significantly upregulated after LPS intervention in vitro by RT-PCR.ConclusionsThe selected key genes Socs3,Il1rn and Ccl7 were up-regulated in myocardial macrophages of septic mice.Socs3,Il1rn and Ccl7 are expected to become new targets for the diagnosis and treatment of sepsis cardiac injury.