基于小鼠脾脏转录组测序探索脓毒症预后关键基因  

Exploring key genes for prognosis of spesis based on transcriptome sequencing of mouse spleen

作　　者：罗福龙 张雨婷 余亚义 胡迎春 陈睦虎 钟武 LUO Fulong;ZHANG Yuting;YU Yayi;HU Yingchun;CHEN Muhu;ZHONG Wu(Emergency Department,the Affiliated Hospital of Southwest Medical University,Luzhou 646000,China)

机构地区：[1]西南医科大学附属医院急诊科,泸州646000

出　　处：《中国免疫学杂志》2024年第4期698-704,713,共8页Chinese Journal of Immunology

基　　金：四川省科技厅项目(2019JDPT0003,2020YFS0517);泸州市-西南医科大学联合课题(2019LZXNYDJ32)。

摘　　要：目的:运用脾脏高通量测序结合生物信息学筛选脓毒症死亡小鼠的关键差异表达基因(DEGs)。方法:①腹腔注射脂多糖(LPS)建立小鼠脓毒症模型,绘制7 d生存曲线确定生存组、死亡组造模剂量。②ELISA验证对照组、生存组、死亡组小鼠外周血浆TNF-α、IL-1β、IL-6、IL-10表达。③使用脓毒症生存组和死亡组脾脏测序,结合生物信息学分析DEGs,筛选关键基因。④RT-PCR及Western blot验证关键基因及蛋白表达差异。结果:①脓毒症生存组造模剂量为15 mg/kg LPS(死亡率为30%),死亡组造模剂量为30 mg/kg LPS(死亡率为80%)。②脓毒症小鼠外周血IL-6、TNF-α、IL-1β表达量较对照组明显升高,IL-10表达水平降低(P<0.05);脓毒症模型组间进行比较,死亡组促炎因子水平较生存组升高,IL-10较生存组降低(P<0.05)。③共筛选出生存组和死亡组中2999个DEGs,其中1185个基因上调,1814个基因下调,筛选出“造血细胞谱系”“原发性免疫缺陷”“非洲锥虫病”“利什曼病”“B细胞受体信号通路”为排名前5的DEGs富集通路,Ifit1、Ifit3、Mx1为筛选出的3个关键基因。④与生存组相比,Ifit1、Ifit3、Mx1在死亡组脾脏组织中的基因及蛋白水平表达均下调(P<0.05)。结论:通过高通量测序与生物信息学筛选出与脓毒症死亡预后相关的关键基因为Ifit1、Ifit3和Mx1,其可能通过病毒感染相关免疫机制影响脓毒症预后。Objective:To screen key differentially expressed genes(DEGs)in dead mice with sepsis by spleen high-throughput sequencing combined with bioinformatics.Methods:①A mouse sepsis model was set up by intraperitoneal injection of lipopolysaccharide(LPS),a 7-day survival curve of mice was drawn,and the modeling doses of survival group and death group were screened out.②Expressions of TNF-α,IL-1β,IL-6 and IL-10 in peripheral blood of mice in control group,survival group and death group were verified by ELISA.③High-throughput sequencing was conducted on spleens of survival group and death group,and the key genes were screened by bioinformatics analysis of DEGs.④Expressions of key genes and proteins were detected by RT-PCR and Western blot.Results:①LPS dosage in survival group was 15 mg/kg(with a mortality of 30%),and LPS dosage in death group was 30 mg/kg(with a mortality of 80%).②Expression levels of IL-6,TNF-αand IL-1βin sepsis mice were significantly higher than those of control group,while expression level of IL-10 was decreased(P<0.05).Comparison of sepsis model groups showed that levels of pro-inflammatory factors in death group were higher than those in survival group,while level of IL-10 was lower than that in survival group(P<0.05).③A total of 2999 DEGs in survival group and death group were screened out by bioinformatics,among which 1185 genes were up-regulated and 1814 genes were down-regulated.Top 5 DEGs enrichment pathways were screened out:"hematopoietic cell lineage""primary immunodeficiency""African trypanosomiasis""leishmaniasis"and"B-cell receptor signaling pathway".Ifit1,Ifit3 and Mx1 were three key genes that were screened out.④Compared with survival group,expressions of genes and proteins of Ifit1,Ifit3 and Mx1 were down-regulated in spleen tissues of the death group(P<0.05).Conclusion:By high-throughput sequencing and bioinformatics,Ifit1,Ifit3 and Mx1 are screened out as key genes related to the death outcome of sepsis,which probably influence the outcome of sepsis through the im

关 键 词：脓毒症 脾脏 转录组学 预后 关键基因 

分 类 号：R392.32[医药卫生—免疫学]