摘要
背景急性肺损伤(acute lung injury,ALI)/急性呼吸窘迫综合征(acute respiratory distress syndrome,ARDS)是危重症医学和基础医学研究重要的关注焦点。明确ALI时关键损伤机制及差异性基因的变化,对探究ALI的发生发展至关重要。目的研究气管内雾化脂多糖(lipopolysaccharides,LPS)致ALI小鼠肺组织的转录组基因谱变化,探索ALI潜在损伤机制及治疗靶点。方法36只小鼠随机分为对照组和气管内雾化LPS组(时间分为致伤后6 h、12 h、24 h,每组各9只)。实验组给予气管内雾化50μL的LPS 15 mg/kg,对照组给予气管内雾化等体积0.9%氯化钠注射液。比较组间组织病理学、病理损伤评分及肺系数变化,检测肺泡灌洗液(bronchoalveolar lavage fluid,BALF)及血清中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)、单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)等水平。肺组织行转录组测序,寻找差异基因,并对上调、下调基因分别进行GO和KEGG富集分析,对持续性改变的差异基因进行GO功能富集整合及共表达网络分析。结果与对照组相比,吸入气管内雾化LPS后小鼠肺组织炎症细胞浸润、水肿加重伴出血,肺损伤评分及肺系数评分逐渐增高,24 h时损伤最严重;TNF-α、IL-6、MCP-1的表达均显著升高(P<0.01),BALF与血浆中表达存在一定差异;转录组学提示12 h组上调基因1545个、下调基因1670个,24 h组上调基因1312个、下调基因1139个。KEGG富集分析显示差异基因涉及炎症反应、遗传信息调控、信号转导等通路,GO富集分析显示差异基因集中在细胞因子生成及免疫反应调控、白细胞的免疫介导及免疫调节、白细胞迁移(上调基因),以及血液循环、信号传导、神经突触、肌肉收缩等(下调基因)。PPI分析提示持续性上调的关键基因包括Tnf、IL-1β、Ifn-γ、Ccl2、Ccl5、Nod2、Tlr2、Lgals9。而持续性下调的关键基因包括Cav1、Sulf1、Sox4、Tgf、Apoe、Tek。结论气管内雾化LPS 12 h建立稳定、可靠的小鼠急性肺损伤模型。Tnf、IL-1β、Ifn-γ、Ccl2、Ccl5、Nod2、Tlr2、Lgals9等关键基因可能是ALI中免疫与炎症反应中的关键调节因子和靶基因。
Background Acute lung injury(ALI)/acute respiratory distress syndrome(ARDS)is an important focus of research in critical care medicine and basic medical science.Clarification of the key damage mechanisms and differential gene changes in ALI is essential to control the development of ALI and explore effective treatments.Objective To investigate the transcriptional profile of lung histology and lung tissue at different time points in mice model of intratracheal aerosolized lipopolysaccharides(LPS)-induced ALI,and explore the potential mechanisms of injury and therapeutic targets of ALI.Methods Thirty-six mice were randomly assigned to the control group and the intratracheal nebulized LPS groups(6 h,12 h and 24 h post-injury,respectively;9 mice in each group).Mice in the experimental group were intratracheally nebulized with 50μL of LPS 15 mg/kg,and the control group were intratracheally nebulized with an equal volume of 0.9%sodium chloride.The histological,pathological injury and lung coefficient changes were compared among the groups,and the levels of TNF-α,IL-6 and MCP-1 in alveolar lavage fluid and serum were detected.Lung tissue transcriptome sequencing was performed in each group of mice to search for differential genes,and GO and KEGG enrichment analyses were performed for up-and down-regulated genes.Then GO functional enrichment integration and co-expression network analyses were performed for differential genes with persistent alterations.Results Compared with the control group,lung histology in the LPS group showed increased inflammatory cell infiltration,and pulmonary vascular edema with a small amount of hemorrhage.The lung injury score and lung coefficient score gradually increased and appeared the most serious injury at 24 h.The expression levels of TNF-α,IL-6,and MCP-1 increased significantly,and there was a certain difference between the expression in BALF and plasma(P<0.01);Transcriptomics suggested that there were 1550 up-regulated genes and 1670 downregulated genes in LPS 12h group,while 1312 up-regulated genes and 1139 down-regulated genes in LPS 24 h group.KEGG enrichment analysis showed that the differential genes were involved in inflammatory response,regulation of genetic information,signal transduction and other pathways.GO enrichment analysis showed that the differential genes were concentrated in the regulation of cytokine production,immune response,leukocyte immune-mediated,immune regulation and leukocyte migration(up-regulated genes),while blood circulation,signaling,nerve synapses,and muscle contraction(down-regulated genes).PPI analysis suggested that key genes persistently up-regulated genes included Tnf,IL-1β,Ifn-γ,Ccl2,Ccl5,Nod2,Tlr2,Lgals9,while key genes persistently down-regulated included Cav1,Sulf1,Sox4,Tgf-β,Apoe,Tek.Conclusion We have established a stable and reliable mouse model of intratracheal nebulization LPS 12 h induced acute lung injury.Tnf,IL-1β,Ifn-γ,Ccl2,Ccl5,Nod2,Tlr2,Lgals9 may be key regulators and target genes in the immune and inflammatory response in ALI.
作者
王佳新
徐耀迪
郑鑫
陈旭昕
张春阳
王凡
丁毅伟
肖漓
韩志海
WANG Jiaxin;XU Yaodi;ZHENG Xin;CHEN Xuxin;ZHANG Chunyang;WANG Fan;DING Yiwei;XIAO Li;HAN Zhihai(Graduate School,Chinese PLA General Hospital,Beijing 100853,China;Department of Pulmonary and Critical Care Medicine,the Sixth Medical Center,Chinese PLA General Hospital,Beijing 100048,China;Institute of Respiratory and Critical Medicine,the Eighth Medical Center,Chinese PLA General Hospital,Beijing Key Laboratory of OTIR,Beijing 100091,China)
出处
《解放军医学院学报》
CAS
北大核心
2023年第6期668-677,共10页
Academic Journal of Chinese PLA Medical School
基金
军队后勤科研项目(CLB20J019
BHJ16J011)。
关键词
急性肺损伤
转录组
气管内雾化
差异性基因
小鼠动物模型
acute lung injury
transcriptome
intratracheal aerosol
differentially expressed genes
mouse animal model
