模拟失重后骨髓来源巨噬细胞转录组高通量测序及分析

High-throughput sequencing and analysis of the transcriptome of bone marrow derived macrophages after simulated weightlessness

目的探索模拟失重对小鼠骨髓来源巨噬细胞基因表达的影响。方法采用尾悬吊法构建模拟失重小鼠模型,对照组不做尾吊处理。模拟失重28 d后提取小鼠骨髓来源巨噬细胞并将其分别诱导为M0型和M1型巨噬细胞。随后,提取细胞总RNA进行转录组测序及分析,并通过实时定量RT-PCR验证重要基因的差异表达。结果转录组测序结果显示,模拟失重组与对照组之间的M0型巨噬细胞共有807个基因差异表达;模拟失重组与对照组之间的M1型巨噬细胞共有898个基因差异表达。GO分析表明,M0型巨噬细胞差异表达基因主要集中于免疫应答、趋化等生物学过程中;M1型巨噬细胞差异基因主要富集于炎症反应、细胞迁移的正调控和单核细胞趋化等生物学过程中。KEGG通路分析发现M0型巨噬细胞的差异基因主要涉及趋化因子、细胞因子-细胞因子受体相互作用等信号通路;M1型巨噬细胞的差异基因主要涉及造血细胞谱系、流体剪切应力等信号通路。进一步分析发现,尾吊组与对照组在M0或M1型巨噬细胞中的差异基因均在细胞因子-细胞因子受体相互作用信号通路中富集,其中尾吊组的M0与M1型巨噬细胞中调节单核细胞/巨噬细胞迁移和浸润的关键趋化因子Ccl2均显著高表达,并进一步通过RT-PCR实验得到验证。结论模拟失重可显著影响小鼠巨噬细胞中以Ccl2为代表的与炎症发生和加重密切相关的基因表达水平,这些改变的基因富集于多个生物学过程,可能对巨噬细胞的黏附、迁移等功能产生影响。

This study was designed to explore the effect of simulated weightlessness on gene expression of mouse bone marrow-derived macrophages.Tail suspension method was used to construct a simulated weightless mouse model,and the tail of the control group was not treated.After 28 days of simulated weightlessness,mouse bone marrow derived macrophages were extracted and induced into M0 and M1 macrophages,respectively.Total RNA of cells was extracted for transcriptome sequencing to screen differential genes,and then gene ontology(GO)and KEGG enrichment analysis were performed for the differentially expressed genes.Data showed that total of 807 genes were differentially expressed in M0 macrophages between the simulated weightlessness group and the control group;a total of 898 genes were differentially expressed in M1 macrophages between the two groups.Moreover,the GO analysis showed that the differential genes in M0 macrophages were mainly enriched in biological processes of immune response and chemotaxis;the differentially expressed genes in M1 macrophages were mainly enriched in biological processes of inflammatory response,positive regulation of cell migration and monocyte chemotaxis.Meanwhile,KEGG pathway analysis revealed that the differentially expressed genes in M0 macrophages were mainly enriched in chemokine signaling pathway and cytokinecytokine receptor interaction;M1 macrophage differentially expressed genes were mainly enriched in hematopoietic cell lineage and fluid shear stress.Further analysis showed that the differentially expressed genes in M0 and M1 macrophages were enriched in the cytokine-cytokine receptor interaction signaling pathway between simulated weightlessness group and control group,and Ccl2,a key chemokine regulating monocyte/macrophage migration and infiltration,was significantly overexpressed in M0 and M1 macrophages in simulated weightlessness group,which was further verified by RT-PCR assay.Therefore,simulated weightlessness can significantly affect the expression level of genes closely related to the occurrence and aggravation of inflammation,represented by CCL2,in mouse macrophages.These altered genes are enriched in multiple biological processes,which may affect the adhesion,migration and other functions of macrophages.