基于转录组测序的创伤早期小鼠腹腔巨噬细胞差异表达基因的分析及验证  

Analysis and validation of differentially expressed genes in post-traumatic mouse peritoneal macrophages based on transcriptome sequencing

作　　者：殷双琴 罗莉 陈涛 梁星河 旷田垠 代伟宏 梁华平 朱俊宇 Yin Shuangqin;Luo Li;Chen Tao;Liang Xinghe;Kuang Tianyin;Dai Weihong;Liang Huaping;Zhu Junyu(State Key Laboratory of Trauma,Burns and Combined Injury,Department of Wound Infection and Drug,Daping Hospital,Army Medical University,Chongqing 400042,China;The 17th Team of Cadet Brigade,College of Basic Medical Sciences,Army Medical University,Chongqing 400038,China;The Second Affiliated Hospital of Hainan Medical University,Haikou 571199,China)

机构地区：[1]陆军军医大学大坪医院战伤感染与特需药品研究室,创伤、烧伤与复合伤国家重点实验室,重庆400042 [2]陆军军医大学基础医学院学员旅17队,重庆400038 [3]海南医学院第二附属医院,海口571199

出　　处：《创伤外科杂志》2023年第1期46-52,55,共8页Journal of Traumatic Surgery

基　　金：国家自然科学基金(81901956);重庆市自然科学基金面上项目(cstc2021jcyj-msxmX0234);国家级大学生创新创业训练计划(202190035010);军事医学前沿创新能力培养计划(2019CXJSC010)。

摘　　要：目的研究严重创伤后小鼠腹腔巨噬细胞基因表达谱的动态变化规律并分析验证其相关生物学功能。方法构建6周左右20~22g的雄性C57BL/6小鼠骨折+失血的严重创伤模型,随机分为对照组和三个严重创伤组(TH2h、TH6h、TH12h),每组10只。在双下肢股骨骨折+眼球40%失血后2、6、12h后提取腹腔巨噬细胞的RNA进行转录组测序。所得序列经质控后,利用差异分析软件DEGseq筛选出差异表达基因,使用Venn分析得到目标差异表达基因集。对目标差异表达基因进行GO富集分析。选择GO中干扰素诱导(interfer on-induced,Ifi)相关的差异表达基因,采用聚类分析这些Ifi相关基因的表达模式,进一步使用定量PCR法检测其mRNA的表达,以验证测序的准确性。结果与对照组相比,TH2h组有903个差异表达基因,TH6h组有1337个差异表达基因,TH12h组有1720个差异表达基因。Venn分析表明,3组中共存在313个差异表达基因,GO功能富集分析发现313个基因主要富集于GO条目下的“生物进程”中,富集程度最大的是“对干扰素β的细胞响应”。选择Ifi相关基因(Ifi35、Ifi202b、Ifi44、Ifit1、Ifi209、Ifit2、Ifit3b、Ifi208、Ifit1bl2、Ifi213、Ifit3、Ifi47、Ifit1bl1、Ifi206、Ifi214)进行聚类分析表明,严重创伤后Ifi相关基因的转录水平均明显下降。定量PCR进一步验证这15个干扰素诱导相关基因的相对表达水平显著下调,与转录组测序的趋势一致(P<0.01)。结论严重创伤后巨噬细胞的基因表达谱发生变化,Ifi相关基因的表达水平显著下调可能是创伤后抗感染能力减弱的一个重要因素,靶向调控Ifi相关基因的表达可作为创伤感染防治的新策略。Objective To study the dynamic changes in gene expression profiles of peritoneal macrophages in mice after severe trauma,and to analyze and verify the related biological functions.Methods A severe trauma model of fracture and blood loss was made on male C57BL/6 mice of 6 weeks old and weighing 20-22g.The mice were divided into control group(Control)and severe trauma groups(TH2h,TH6h,TH12h),with 10 mice in each group.RNA was extracted from peritoneal macrophages before(Control)and 2,6,and 12 h after trauma modeling(femoral fractures of both lower limbs and 40%blood loss),and the transcriptome was sequenced.After the obtained sequences were subjected to quality control,the differentially expressed genes(DEGs)were screened out by the differential analysis software DEGseq,and the target DEGs set was obtained by Venn analysis.GO enrichment analysis was performed on the target DEGs.The interferon induction(Ifi)-related genes in the GO enrichment analysis were selected,the expression patterns of these Ifi-related genes were analyzed by clustering,and the mRNA expression of these genes was further detected by qPCR to verify the accuracy of sequencing.Results Compared with the control group,there were 903 DEGs in the TH2h group,1,337 DEGs in the TH6h group,and 1,720 DEGs in the TH12h group.Venn analysis showed that there were 313 DEGs in common among the three trauma groups.GO functional enrichment analysis found that 313 genes were mainly enriched in the"biological process"under the GO item,and the most enriched GO term was"cellular response to interferon-beta".Ifi-related genes(Ifi202b,Ifi44,Ifit1,Ifi209,Ifit2,Ifit3b,Ifi208,Ifit1bl2,Ifi213,Ifit3,Ifi47,Ifit1bl1,Ifi206,Ifi214)were selected for cluster analysis.The result showed that the transcription levels of Ifi-related genes were significantly decreased after severe trauma.qPCR further verified that the relative mRNA expression levels of these 15 Ifi genes were significantly down-regulated(P<0.01),which was consistent with the trend of transcriptome sequencing.Conclusi

关 键 词：创伤 巨噬细胞 干扰素诱导相关基因 RNA-SEQ 

分 类 号：R318[医药卫生—生物医学工程]