革兰阳性菌及革兰阴性菌感染脓毒症患者外周血单个核细胞关键基因鉴定及共表达网络分析

doi:10.3785/j.issn.1008-9292.2020.12.08

浙江大学学报（医学版）

2020, Vol. 49

Issue (6): 732-742 DOI: 10.3785/j.issn.1008-9292.2020.12.08

原著

革兰阳性菌及革兰阴性菌感染脓毒症患者外周血单个核细胞关键基因鉴定及共表达网络分析

李璐¹(

),方俊君²,李志涛²,沈磊星³,王国彬^2,*(

),傅水桥^2,*(

)

1. 浙江大学医学院附属第一医院临床药学部, 浙江杭州 310003
2. 浙江大学医学院附属第一医院外科重症监护室, 浙江杭州 310003
3. 浙江大学城市学院药学院, 浙江杭州 310015

Master genes and co-expression network analysis in peripheral blood mononuclear cells of patients with gram-positive and gram-negative sepsis

LI Lu¹(

),FANG Junjun²,LI Zhitao²,SHEN Leixing³,WANG Guobin^2,*(

),FU Shuiqiao^2,*(

)

1. Department of Clinical Pharmacy, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
2. Surgical Intensive Care Unit, the First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China
3. School of Pharmaceutical Sciences, Zhejiang University City College, Hangzhou 310015, China

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摘要：

目的: 探讨革兰阳性菌与革兰阴性菌感染脓毒症患者相关的生物标志物。方法: 在美国国立生物技术信息中心平台获取GSE9960表达谱芯片，包含16例非感染全身炎症反应综合征（SIRS）患者外周血单个核细胞（PBMC）样本，17例革兰阳性菌感染脓毒症患者PBMC样本，以及18例革兰阴性菌感染脓毒症患者PBMC样本。利用基因集富集分析和加权基因共表达网络分析探索GSE9960基因表达谱芯片中革兰阳性菌感染与革兰阴性菌感染脓毒症富集功能通路与共表达网络模块，并对高度相关模块进行通路注释。利用R语言分析革兰阳性菌与革兰阴性菌感染脓毒症中差异表达基因，探索差异表达基因蛋白-蛋白相互作用网络。将高度相关模块中的枢纽基因与差异表达基因重叠，确定为主要差异表达基因，并进一步针对主要差异表达基因在临床样本中进行验证。结果: 革兰阳性菌感染脓毒症与革兰阴性菌感染脓毒症间富集通路存在显著差异，非感染SIRS和革兰阴性菌感染脓毒症存在相反的共表达网络；而革兰阳性菌感染和革兰阴性菌感染脓毒症存在完全不同的共表达网络。进一步在临床样本中验证显示，主要差异表达基因TYMS在革兰阳性菌感染脓毒症患者PBMC中显著上调（P < 0.05）；CD3D在革兰阴性菌感染脓毒症中表达下调（P < 0.01），而IRAK3则显著上调（P < 0.05）。结论: 本研究结果有利于加深对不同细菌感染脓毒症发病机制的理解，并为脓毒症诊断、分型及经验性抗菌药物治疗提供潜在生物标志物。

关键词： 脓毒症; 全身炎症反应综合征; 革兰阳性菌; 革兰阴性菌; 外周血单个核细胞

Abstract:

Objective: To investigate the functional pathways enriched and differentially expressed genes (DEGs) in peripheral blood mononuclear cells (PBMCs) of patients with gram-positive and gram-negative sepsis. Methods: Dataset GSE9960 obtained from NCBI GEO database containing PBMC samples from 16 non-infectious systematic inflammatory response syndrome (SIRS) patients, 17 gram-positive septic patients and 18 gram-negative septic patients were included in the study. Functional pathway annotations were conducted by gene set enrichment analysis and weighted gene co-expression network analysis. DEGs were filtered and master DEGs were then validated in PBMCs of gram-positive septic, gram-negative septic and non-infectious SIRS patients. Results: The enriched gene sets in gram-positive sepsis and gram-negative sepsis were significantly different. The results indicated the opposite co-expression networks in SIRS and gram-negative sepsis, and the entirely different co-expression networks in gram-positive and gram-negative sepsis. Furthermore, we validated that TYMS was up-regulated in gram-positive sepsis (P < 0.05), CD3D was down-regulated in gram-negative sepsis (P < 0.01), while IRAK3 was up-regulated in gram-negative sepsis (P < 0.05). Conclusion: The results indicate that there are differences in the mechanism and pathogenesis of gram-positive and gram-negative sepsis, which may provide potential markers for sepsis diagnosis and empirical antimicrobial therapy.

Key words: Sepsis Systemic inflammatory response syndrome Gram-positive bacterium Gram-negative bacterium Peripheral blood mononuclear cell

收稿日期: 2020-08-13 出版日期: 2021-01-14

CLC:

R447

基金资助: 浙江省自然科学基金(LYY20H280004);浙江省医药卫生科技计划(2020381061)

通讯作者: 王国彬,傅水桥 E-mail: lucille@zju.edu.cn;wgb8@163.com;2200048@zju.edu.cn

作者简介: 李璐(1988-), 女, 博士, 主管药师, 主要从事抗感染临床药学研究; E-mail:lucille@zju.edu.cn; https://orcid.org/0000-0003-4573-8538

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引用本文:

李璐,方俊君,李志涛,沈磊星,王国彬,傅水桥. 革兰阳性菌及革兰阴性菌感染脓毒症患者外周血单个核细胞关键基因鉴定及共表达网络分析[J]. 浙江大学学报（医学版）, 2020, 49(6): 732-742.

LI Lu,FANG Junjun,LI Zhitao,SHEN Leixing,WANG Guobin,FU Shuiqiao. Master genes and co-expression network analysis in peripheral blood mononuclear cells of patients with gram-positive and gram-negative sepsis. J Zhejiang Univ (Med Sci), 2020, 49(6): 732-742.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2020.12.08 或 http://www.zjujournals.com/med/CN/Y2020/V49/I6/732

表 1 实时逆转录PCR引物序列

图 1 革兰阳性菌与革兰阴性菌感染脓毒症的基因集富集分析图

图 2 加权基因共表达网络分析革兰阳性菌感染与革兰阴性菌感染脓毒症的共表达网络

图 3 革兰阳性菌和革兰阴性菌感染脓毒症高度相关模块基因本体(GO)和京都基因与基因组百科全书(KEGG)通路富集结果

模块	枢纽基因
棕色模块	SAMD8、TLR6、IFRD1、IL17RA、PCYT1A、PRO2852、FCER1G、RNF149、SLC12A6、FAM160A2、CAMKK2、GALNT3、COL4A3BP、STAT5B、RNASEL、MYO1F、ALPK1、C1RL、FAM126B、VPS37B、TSPO、DENND5A、LIMK2、SLC2A14、PPP1R10、PGK1、DNTTIP1、MKNK1、RNF24、ARL8A、MSL1、ERI1、SDF2、TLR8、GYG1、RP11-295G20.2、ZNF467、LOC101928676、ZBTB34、UBXN2B、OSBPL2、TUBA1A、FAM53C、RBMS1、NBEAL2、HIP1、ARL11、XPO6、CEBPB、TMCO3、CTBS、SELPLG、IL10RB、DYSF、MTF1、GNAQ、GAS7、SLC2A3、KIF1B、CCDC126、IQGAP1、RHOG、IFNAR1、PGM2、STAT3、RNF13、PRKCD、PECR、RGS3、SNX11、SLMAP、NDUFB3、RRAGC、NUPL1、NFIL3、NCF4、MAP3K5、APOBR、STX3、FLOT2、SKAP2、TSEN34、PPP4R1、SLCO3A1、FPR2、CSNK1D、UPP1、CHSY1、MEF2A、HK3、IRAK3、SIPA1、PHF20L1、PLIN3、SLC25A44、RTFDC1、CKLF、ATP6V1C1、EIF4E3、PIK3CG、TLE3、CHMP2A、FPR1、PTBP3、LINC01000、RGS19、NRBF2、ZNF281、TLR1、CHMP2B、ATXN1、GLT1D1、CFLAR、NCF2、LYN、KLF7、THOC5、LIN7A、RIT1、JMJD6、FAM160B1、ARID3A、ARPC2、FAM129A、C17orf62、FLOT1、BNIP2、EXOC6、PGS1、PXK、SVIL、PFKFB4、WDFY3、RALB、H3F3A、HCK、C4orf3、BIN2、CANT1、GNAI3、LPCAT2、LILRB3、LPGAT1、RFWD2、FNDC3B、LCP1、PRR13、TMEM260、BTBD10、GNG5、CD58、SHOC2、CSF3R、GMFG、CAP1、BAZ1A、AGO4、AGPAT9、S100A11、PJA2、RLIM、ALOX5、ZNF438、RARA-AS1、RPS6KA1、OSTF1、TMEM120A、HSDL2、PPP1R12A、HN1、C20orf24、POR、DENND3、CKLF、FRAT2、NIN、CAB39、YIPF1
黄色模块	ZBTB25、GSPT2、NDUFAF4、LINC00263、ZNF84、IL32、CD3G、MAGEH1、ITM2A、SLC4A7、CLEC2D、JMY、TRAF5、NMT2、TRIB2、KPNA5、PLEKHA1、IFFO2、TRMT13、RASGRP1、LPIN1、PTPRCAP、PJA1、CD3D、PRKCQ、THEM4、LYRM7、SIDT1、ZNF32、CD247、DIS3L2、NFATC2、BCL11B、PPP1R16B、ZNF420、TRBC1、NR1D2、ARL4C、EVL、LY9、TRAC、KIF3A、TRAC、ZNF286A、SKAP1、ZAP70、LCK、DYRK2
粉色模块	OAS3、DDX60、IFIT5、HERC5、OASL、XAF1、OAS2、IFI44、ISG15、HERC6、PARP14、DTX3L、IFIT1、APOL6
绿色模块	CDC6、RMI2、WDHD1、WDR34、KIFC1、UBE2C、AUNIP、IQGAP3、KIF18A、BRCA2、CDKN3、KIAA0101、STIL、NCAPG2、GINS3、TRIP13、SKA1、CEBPE、CRNDE、CASC5、ANKRD18A、CEP55、MCM10、KIAA1524、STMN1、GTSE1、ZWINT、LOC81691、UBE2T、PLS1、BUB1、NEK2、AURKA、ASPM、ORC1、ESPL1、AURKB、SUV39H2、HMMR、TYMS、CDC20、CENPE、ZNF788、CCNB1、GINS1、PRC1、BRIP1、SLC27A2、MELK、CDT1、CDCA8、RAD51AP1、TCF19、SHCBP1、UHRF1、MND1、CDC45、DLGAP5、KIF20A、KIF4A、KIF14、POLE2、FANCI、KIF2C、CCNA2、POLQ、CENPA、FOXM1、DTL、CHEK1、TMEM52B、DEPDC1、SPAG5、NUF2、NUSAP1、CDCA2、HJURP、BIRC5、NCAPH、TPX2、NEIL3、E2F8、TTK、TOP2A、KIF11、DEPDC1B、NCAPG、ANLN、MKI67、PLK4、CDCA5、BUB1B、CCNE2、CDK1、SPC25、RAD51、TK1、KIF15、KIF18B、OIP5、CDCA3

表 2 革兰阳性菌感染与革兰阴性菌感染脓毒症共表达网络相关模块中的枢纽基因

表 3 革兰阳性菌感染脓毒症差异表达的4个下调基因和前5个上调基因

表 4 革兰阴性菌感染脓毒症差异表达的前5个上调基因和前5个下调基因

图 4 革兰阳性菌感染和革兰阴性菌感染脓毒症差异表达基因蛋白-蛋白相互作用网路及功能通路注释

图 5 主要差异表达基因在患者外周血单个核细胞中的验证结果

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