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  被引量:1

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

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作  者:胡东霞[1] 陈盛松[2] 余韵[1] 胡龙龙 刘亮 余玲玲[1] Hu Dongxia;Chen Shengsong;Yu Yun;Hu Longlong;Liu Liang;Yu Lingling(Department of Rehabilitation,The Second Affiliated Hospital of Nanchang University,Nanchang 330006,China;Department of Respiratory and Critical Care,Jiangxi Provincial People's Hospital,Nanchang 330006,China;Department of Cardiology,The Second Affiliated Hospital of Nanchang University,Nanchang 330006,China)

机构地区:[1]南昌大学第二附属医院康复医学科,南昌330006 [2]江西省人民医院呼吸与危重症学科,南昌330006 [3]南昌大学第二附属医院心血管内科,南昌330006

出  处:《中华心血管病杂志》2023年第7期759-768,共10页Chinese Journal of Cardiology

基  金:国家自然科学基金(82100308);江西省自然科学基金(20202BABL216035,20202BAB206042);江西省教育厅科技计划(GJJ200101);江西省中医药管理局科技计划(2019A067)。

摘  要: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�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 regula

关 键 词:巨噬细胞 脓毒症心肌损伤 细胞因子信号转导抑制因子3 白细胞介素1受体拮抗剂 CC趋化因子7 

分 类 号:R459.7[医药卫生—急诊医学] Q811.4[医药卫生—治疗学]

 

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