基于RNA-seq技术分析活性氧对未成熟树突状细胞迁移能力的促进作用  

Analysis of the Promotion with Reactive Oxygen Species on Migration of Immature Dendritic Cells Based on RNA-seq Technique

作　　者：董美玲 罗赵雅靖 吴仪 陈晋 岳萍 胡祖权 曾柱 王赟 DONG Meiling;LUO Zhaoyajing;WU Yi;CHEN Jin;YUE Ping;HU Zuquan;ZENG Zhu l;WANG Yunl(Engineering Center of Cellular Immunotherapy of Guizhou Province,School of Basic Medical Sciences,Guizhou Medical University,Guiyang,550000;Immune Cells and Antibody Engineering Research Center in University of Guizhou Province,School of Biology and Engineering(School of Modern Industry for Health and Medicine),Guizhou Medical University,Guiyang,550)

机构地区：[1]贵州医科大学基础医学院,贵州省细胞免疫治疗工程研究中心,贵阳550000 [2]贵州医科大学生物与工程学院(健康医药现代产业学院),贵州省普通高等学校免疫细胞与抗体工程研究中心,贵阳550000

出　　处：《基因组学与应用生物学》2024年第11期1859-1871,共13页Genomics and Applied Biology

基　　金：国家自然科学基金项目(12202111、12132006);贵州省科技计划项目(黔科合基础ZK[2022]一般385、黔科合基础ZK[2022]一般397、黔科合平台人才[2021]5637号);贵州医科大学基金项目(21NSFCP35、校博合J字[2021]042号)共同资助。

摘　　要：本研究在检测未成熟树突状细胞(immature dendritic cells,imDCs)响应胞外活性氧(reactive oxygen species,ROS)后的细胞周期和迁移能力基础上,通过RNA测序(RNA sequencing,RNA-seq)技术分析差异表达基因(differentially expressed genes,DEGs),探索ROS促进imDCs迁移的潜在机制。利用H_(2)O_(2)模拟ROS构建imDCs氧化应激模型,利用CCK8法检测细胞活力,采用流式细胞术检测细胞凋亡和细胞周期变化,利用细胞实时成像实验分析imDCs的自由迁移情况。提取总RNA并进行RNA-seq,对DEGs进行GO和KEGG富集分析,按照DEGs表达变化|log_(2)FC|且FDR≤0.01筛选代谢及骨架调节相关转录基因。结果表明,H_(2)O_(2)处理浓度小于100μmol/L时,imDCs的细胞活力和凋亡均无显著变化;imDCs在ROS环境中的细胞周期停滞在G0/G1期,且细胞的自由迁移能力增强。RNA-seq结果显示,H_(2)O_(2)处理组与对照组相比,共有282个DEGs,其中225个基因表达下调,57个基因表达上调。GO和KEGG分析发现这些基因参与了代谢、细胞周期和细胞骨架等相关通路,其中与异源物质代谢相关的Gsta1、Gsta3、Cstdc5和Prdx1等基因表达上调,Shmt2基因表达下调;参与骨架调节的S100a8、Ppbp和Tm4sf19等基因表达上调,而Nusap1、Stmn1、Kif20a和Prc1等基因表达下调。本研究表明,在imDCs响应胞外ROS过程中抗氧化系统激活、细胞骨架重塑与细胞周期停滞可能参与调控其迁移能力。Based on the detection of the cell cycle and migration ability of immature dendritic cells(imDCs)in response to reactive oxygen species(ROS),this study aims to analyze difrentially expressed genes(DEGs)through RNA sequencing(RNA-seq)technology and explore the potential mechanism of ROS promoting the migration of imDCs.The oxidative stress model of imDCs was constructed by simulating ROS with H_(2)O_(2).The CCK8 method was used to detect cell viability.The flow cytometry was deployed to analyze cell apoptosis and the changes of cell cycle.The free migration of imDCs was analyzed by cell real-time imaging.Total RNA was extracted and RNA-seq was performed.GO and KEGG enrichment analysis of DEGs was performed.Transcripts related to metabolism and skeleton regulation were screened according to the changes of DEGs expression |log_(2)FC|≥1 and FDR≤0.01.The result showed that there was no significant changes in cell viability and apoptosis of imDCs when H_(2)O_(2) concentration was less than 100μmol/L.The cell cycle of imDCs was arrested at GO/GI phase and the free migration ability of imDCs was enhanced when in the ROS environment.RNA-seq data showed there were 282 DEGs between the control group and the H_(2)O_(2)treatment group.Among them,225 differentially expressed genes were down-regulated,and 57 were up-regulated.GO and KEGG enrichment analyses found that these genes were involved in metabolism,cell cycle and cytoskeleton-related signalling pathways.Gstal,Gsta3,Cstdc5 and Prdxl genes involved in response to xenobiotic metabolism were up-regulated but Shmt2 was down-regulated.S100a8,Ppbp and Tm4sf19 genes involved in skeleton regulation were also up-regulated.Simultaneously,Nusapl,Stmnl,Kif20 and Prcl were down-regulated.Therefore,the activation of the antioxidant system,cytoskeletal remodeling and cell cycle arrest may be involved in regulating the migration ability of imDCs in response to extracellular ROS.

关 键 词：活性氧 树突状细胞 氧化应激 细胞骨架 细胞迁移 

分 类 号：R392.12[医药卫生—免疫学]